GECKIL Genetik Mühendisliği Laboratuarı

The Vitreoscilla hemoglobin

(# 1 modulator in metabolic engineering)

The Vitreoscilla hemoglobin (commonly abbreviated as VHb) was first discovered by Dale A. Webster in 1986 (1). However, Dale (latter to be my co-advisor together with Benjamin C. Stark, both at IIT, Chicago) originaly discovered it in 1966 (2) and given its prevalent presence in the cytoplasm but not in the membrane fraction, he named it as "the soluble form of the cytochrome bo", the terminal oxidase of bacterial respiratory chain.

I had the privilege to be one of the firsts to study the VHb and its gene (vgb) with Dale and Ben. Ben has also been known for his discovery of "catlytic RNA" during his postdoc study at Yale University, securing the Nobel Prize in Chemistry in 1989 for his advisor Sydney Altman. Having the opportunity to work with such high-caliber scientists was the most revarding part of my life with science. Robert M. Roth, then the chairman of the Biology Department, was another mentor whom I liked how he orchestrated his teaching with an explicit Brooklyn accent. Thank to all three for believing in me, a naive Turkish native, to be their TA for their courses (Advanced Biochemistry by Dale, Molecular Cell Biology by Ben, and Microbial Genetics and Genetic Engineering by Bob).

For sometime, the hemoglobins were considered to be the proteins of only eukaryotic origin. The main reason for this view is that, most eukaryotic organisms (with the exeption of the single celled ones) are multicellular and the free diffusion of oxygen into cells deep in the thick tissues and organs would not be possible without a suitable carrier (i.e. hemoglobin). Now, we know that hemoglobins exisit virtually in all kingdoms of life. But, what role such proteins may play in free living single cells, such as bacteria, which acquire oxygen from the environment readily through passive diffusion. Although, this is a question of ongoing debate, we now also know that not only do these ubiquitous protiens transport the oxygen between tissues, the known function of hemoglobins, but also they play other roles ranging from intracellular oxygen transport to catalysis of redox reactions.

Aerobic (oxygen using) organisms started to dominate the life on earth about 3 billion years ago, as the anoxic (oxgen-free) atmosphere become more and more oxic (oxygenated) with the advent of photosynthesis about 4 billion years ago. With some minor exeptions (e.g., anerobic bacteria), today life on earth in all levels, as we know it, is oxygen dependent and it is no surprise that many genes (if not all) in all these life forms from bacteria to human are directly or inderectly regulated by oxygen. Moreover, oxygen is the final electron acceptor in the respiratory chain which is coupled to oxidative phosphorylation through which all organisms generate about 90 % of thier energy (i.e., ATP). So without this gas, i.e., oxygen, life as a whole would be succumed to death.

In multicellular organsims with bulky bodies, oxygen transfer into the cells residing far from perifery would be a problem if not solved by a specilized carrier. Because, oxyen is easly disolved in cellular and extracellular milieu where many oxygen hungry molecules or reactions can readly consume it, hindering its further transfer. Life solved this problem by creating hemoglobins and veins. The tracks, that are veins, provide blood cells (i.e. erythrocytes) which carry oxygen loaded hemoglobin to their destination all over the body (when there is no gain to oxygen, ther is pain!). With its highly efficient oxyen-carbon dioxyde affinity kinetics, hemoglobin relaeses more oxygen to regions where there is much need (low oxygen, high carbondioxide regions).

A bacterium is both an organism and also a cell. It is not in tissue form and does not have extensive intracellular structures (i.e., organeles). As for the cells in organisms, oxygen is easly diffuesed through its membrane and the final destinations are not that far when its size is considered (~1 x 2 µm). So, why invest so much energy to synthesize a 146 amino asit long hemoglobin protein (who knows how many molecules of it).

The term "metabolic engineering" was first coined by late James E. Bailey (then at Caltech) and Gregory Stephanopoulos (Massachusetts Institute of Technology (MIT)) in 1991 in the same issue of the journal Science (3,4). Prof. Bailey was also a pioneer in VHb studies (and his group was a rival of our laboratory at IIT) and Prof. Stephanopoulos is the editor-in-chief of the journal Metabolic Engineering.

GEÇKİL Genetik Mühendisliği Laboratuarı biyokimya, genetik ve moleküler biyoloji tekniklerini kullanarak içinde bulunduğumuz bir araştırmacı grubu tarafından keşfedilen ve ilk prokaryotik hemoglobin olan Vitreoscilla hemoglobininin işlevi ve metabolik mühendislikteki potansiyel kullanımını çalışmaktadır. Araştırmamızın amacı, oksijen taşıyan böyle bir proteinin doğal konakçısındaki işlevini aydınlatmak ve genleri oksijenle regüle olan endüstriyel öneme sahip birçok ürünün üretiminde bir metabolik modülatör olarak kullanılabilecek bu proteinin potansiyelini ortaya koymaktır. Kanser kemoterapisinde kullanılan bir enzim olan "asparaginaz", Alzheimer's ve Parkinson hastalıklarında kullanılan ilaçlar olan "dopa" and "dopamin" bu ürünlerin başlıcalarıdır. Laboratuarımızın yeni çabalarından biri, tüm kanserlerin 10 ana belirtecinden biri sayılan ve Warburg etkisi olarak da bilinen "aerobik glikoliz"de mTOR sinyal kompleksinin rolünü anlamaktır.

ARAŞTIRMA ALANI

Vitreoscilla hemoglobininin genetik mühendisliği

  • Vitreoscilla hemoglobin geninin çeşitli endüstriyel mikroorganizmalara klonlanamsı, ,izolasyonu ve eksprasyonu
  • Vitreoscilla hemoglobinin genetik mühendisliği ve klonlanmış bakterilerin oksidative stres ve antioksidan özellikleri©
  • Vitreoscilla hemoglobinin biyoremediasyonda kullanım potansiyeli
  • Vitreoscilla hemoglobini kullanarak endüstriyel önemi olan çeşitli fermentatif solventlerin üretimi
  • Vitreoscilla hemoglobini kullanarak bakteriyel orijinli antilösemik asparaginazın üretiminin arttırılması©

Metabolik Yeniden Programla ve Kanser

  • İnsan kanserlerinde mTOR sinyal yolağının rolü
  • Ghrelin ve Hücre Fizyolojisi

Biyolojik proses mühendisliği

  • Endüstriyel değeri büyük ürünlerin biyoprosesi için metot geliştirme (ör. kanser kemoterapisinde kullanılan enzimler

1. Wakabayashi, S.; Matsubara, H.; Webster, D. A., Primary Sequence of a Dimeric Bacterial Hemoglobin from Vitreoscilla. Nature 1986, 322, (6078), 481-483.

2. Webster, D. A.; Hackett, D. P., The purification and properties of cytochrome o from Vitreoscilla. J Biol Chem 1966, 241, 3308-3315.

3. Bailey, J. E., Toward a Science of Metabolic Engineering. Science 1991, 252, (5013), 1668-1675.

4. Stephanopoulos, G.; Vallino, J. J., Network Rigidity and Metabolic Engineering in Metabolite Overproduction. Science 1991, 252, (5013), 1675-1681.

Tree of Life

DERSLER

  • 00301/00302 Biyokimya I ve II
  • 00351/00352 Biyokimya Lab I ve II
  • 00469 Biyokimyasal Teknikler (seçmeli)
  • 00581 Biyolojide Analitik Metotlar
  • 00582 Genetik Mühendislik Uygulamaları
  • 00668 Genetik Mühendislik
  • 00695 Moleküler Biyoloji ve Biyokimyada Pratik Uygulamalar


P-28: Demirci U and Geckil H.
Editorial: Micro and nanofluidics – applications in biotechnology. 
Biotechnology Journal, 2011; 6(2): 131
 
P-27: Gurkan UA, Moon S, Geckil H, Xu F, Wang S, Lu TJ and Demirci U.
Miniaturized lensless imaging systems for cell and microorganism visualization in point-of-care testing. 
Biotechnology Journal, 2011; 6(2): 138-149.
 
 
 
 
 

 

Issue Editors:Utkan Demirci (Harvard Üniversitesi) and Hikmet Geckil

 
P-26: Geckil H, Xu F, Xiaohui Z, Moon S-J, Demirci U.
Engineering hydrogels as extracellular matrix mimics.
Nanomedicine, 2010; 5(3): 469-484.
 
P-25: Geckil H and Calik P. 
Editorial: Biotech in Turkey. 
Biotechnology Journal, 2009 4(7), 951.
 
P-24: Kurt AG , Aytan E, Ozer U, Ates B and Geckil H
Production of L-DOPA and dopamine in bacteria bearing Vitreoscilla hemoglobin gene.
Biotechnology Journal2009 4(7), 1077-1088.
Issue Editors
Hikmet Geckil and Pinar Calik (Orta Doğu Teknik Üniversitesi)
P-23: Aydin S, Karatas F and Geckil H.
Simultaneous Quantification of Acylated and Desacylated Ghrelin in Biological Fluids.
Biomedical Chromatography, 20008; 22(12): 1354-1359.

P-22: Aydin S, Geckil H, Kilic N, Erman F, Kilic SS and Yesilada O.
Is Ghrelin a Natural Anti-Microbial Agent?
Turkish Journal of Medical Sciences, 2008; 38(2): 187-187.

P-21: Aydin S, Geckil H, Karatas F, Donder E, Kumru S, Kavak EC, Colak R, Ozkan Y, Sahin I.
Milk and blood ghrelin level in diabetics.
Nutrition, 2007; 23(11-12): 807-811.

P-20: Aydin S, Ozercan IH, Geckil H, Dagli F, Aydin S, Kumru S, Kiliç N, Sahin I, Ozercan MR.
Ghrelin is present in teeth.
Journal of Biochemistry and Molecular Biology, (2007); 40(3):368-372.

P-19: Aydin S, Geckil H, Karaoglu A, Elkiran ET.
Ghrelin: A novel peptide with therapeutic effect in certain cancers?
Medical Hypotheses, (2007); 69(5): 1157-1158.

P-18: Geckil H, Gencer S, Ates B, Ozer U, Uckun M, Yilmaz I.
Effect of Vitreoscilla hemoglobin on production of a chemotherapeutic enzyme, L-asparaginase, by Pseudomonas aeruginosa.
Biotechnology Journal, (2006); 1(2): 203-208.
 
P-17: Aydin S, Ozercan HI, Aydin S, Ozkan Y, Dagli F, Oguzoncul F, Geckil H.
Biological rhythm of saliva ghrelin in humans.
Biological Rhythm Research, (2006); 37(2): 169-177.
 
P-16: Aydin S, Geckil H, Zengin F, Ozercan HI, Karatas F, Aydin S, Balik DT, Ozkan Y, Dagli F, Celik V.
Ghrelin in plants: What is the function of an appetite hormone in plants?
Peptides, (2006); 27(7): 1597-1602.
 
P-15: Kahraman H and Geckil H.
Degradation of benzene, toluene and xylene by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene.
Engineering in Life Sciences, (2005); 5(4): 363-368.
 
P-14: Geckil H, Ates B, Durmaz G, Erdogan S, Yilmaz I.
Antioxidant, free radical scavenginging and metal chelating characteristics of propolis.
American Journal of Biochmemistry and Biotechnology, 2005; 1(1): 27-31.
 
P-13: Geckil H, Ates B, Gencer S, Uckun M, and Yilmaz I.
Membrane permeabilization of Gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of L-asparaginase: an enzyme used in cancer therapy.
Process Biochemistry, 2005; 40(2): 573-579.
 
P-12: Geckil H, Barak Z, Chipman DM, Erenler SO, Webster DA and Stark BC.
Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene.
Bioprocess and Biosystems Engineering, 2004; 26(5): 325-330.

P-11: Geckil H, Arman A, Gencer G, Ates B and Yilmaz HR.
Vitreoscilla hemoglobin renders Enterobacter aerogenes highly susceptible to heavy metals.
Biometals, 2004; 17(6): 715-723.

P-10: Geckil H, Gencer S and Uckun M.
Vitreoscilla hemoglobin expressing Enterobacter aerogenes and Pseudomonas aeruginosa respond differently to carbon catabolite and oxygen repression for production of L-asparaginase: an enzyme used in cancer therapy.
Enzyme and Microbial Technology, 2004; 35(2-3): 182-189.

P-09: Ates B, Yilmaz I, Geckil H, Iraz M, Birincioglu M, Fiskin K.
Protective role of melatonin given either before ischemia or prior to reperfusion on intestinal ischemia-reperfusion damage.
Journal of Pineal Research, 2004; 37(3): 149-152.

P-08: Geckil H and Gencer S.
Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake.
Applied Microbiology and Biotechnology, 2004; 63(6): 691-697.

P-07: Erenler SO, Gencer S, Geckil H, Stark BC, and Webster DA.
Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth an oxygen uptake.
Applied Biochemistry and Microbiology, 2004; 40(3): 288-295.

P-06: Geckil H, Gencer S, Kahraman H, and Erenler SO.
Genetic engineering of Enterobacter aerogenes with Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress.
Research in Microbiology, 2003; 154(6): 425-431.

P-05: Munzuroglu *, Karatas F, and Geckil H.
The vitamin and selenium contents of apricot fruit of different varieties cultivated in different geographical regions.
Food Chemistry, 2003; 83(2): 205-212.

P-04: Munzuroglu O, Obek E, and Geckil H.
Effects of simulated acid rain on the pollen germination and pollen tube growth of apple (Malus sylvestris Miller cv. Golden).
Acta Biologica Hungarica, 2003; 54(1): 5-103.

P-03: Yurekli F, Geckil H, and Topcuoglu F.
The synthesis of indole-3-acetic acid by the industrially important white-rot fungus Lentinus sajor-caju under different culture conditions.
Mycological Research, 2003; 107(3): 305-309.

P-02: Munzuroglu O and Geckil H.
Effects of metals on seed germination, root elongation, coleoptile and hypocotyl growth in Triticum aestivum and Cucumis sativus.
Archives of Environmental Contamination and Toxicology, 2002; 43(2): 203-213.

P-01: Geckil H, Stark BC, and Webster DA.
Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently affected by the genetically engineered Vitreoscilla hemoglobin gene.
Journal of Biotechnology, 2001; 85(1): 57-66. 
 
PUBLISHED CONFERENCE PROCEEDINGS
Icen I, Celik V, Pelle R, Aytan E, Munzuroglu O, Geckil H.
Isolation, cloning and sequence analysis of gene encoding the lactate dehydrogenase enzyme from Theileria parva.                            
FEBS JOURNAL, 2011, 278, 329.
 
Celik V, Icen I, Pelle R, Kurt AG, Munzuroglu O, Geckil H.
A new candidate for antitheilerial target: isolation, cloning and sequence analysis of gene encoding the enolase enzyme from Theileria parva.
FEBS JOURNAL, 2011, 278, 329.
 
Erenler S and Geckil H.
Cloning, isolation and expression of l-asparaginase gene (ansB) in different gram-negative bacteria expressing Vitreoscilla hemoglobin 
 
Celik V, Icen I, Pele P, Munzuroglu O, Geckil H.
Expression of gene encoding Theileria parva enolase in Escherichia coli JM103 strain 
 
Ates B, Gencer S, Erenler SO, Uckun M, Ozer U, Yilmaz I, and Geckil H.
Production of L-asparaginase, a chemotherapeutic enzyme, in bacteria expressing Vitreoscilla haemoglobin.
FEBS JOURNAL, (2006); 273: 139-139.
 
Kahraman H, Gencer S, Geckil H.
Effect of Vitreoscillaon L-lysine alpha-oxidase, a chemotherapeutic enzyme, from Pseudomonas aeruginosa.
FEBS JOURNAL, (2006); 273: 139-140.
 
PUBLICATIONS in TURKISH LANGUAGE
A-01: Aydin S, Geckil H, Caylak E and Kilic N (2004). Mikroorganizmalarin kanser tedavisinde kullanimi.
Firat Medical Journal, 9(2): 30-34.
 
A-02: Kahraman H and Geckil H* (2005). Benzoik Asitin Vitreoscilla hemoglobin geni aktarılmış Pseudomonas aeruginosa tarafından yıkımı.
Fen ve Muhendislik Bilimleri Dergisi, Firat Universitesi, 17(2); 342-348.
 
A-03: Geckil H (2005). "Iki Emekli Bilim Adaminin Hikayesi: Genetige Karsi Biyokimya".
 
A-04: Geckil H (2006). "Molekuler Yasam Bilimlerinde Ph.D. Derecesi Icin Standartlar" (Uluslararasi Biyokimya ve Molekuler Biyoloji Birliği (IUBMB)'nin "Standarts for the Ph.D. Degree in the Molecular Biosciences" isimli orijinal calismasindan yapilmiş Turkce tercume). 
This copyright free study has been published or publicized in: 
A-05: Hikmet Geçkil. Genomlarla Eğlence: Mikoçiğner DNA Bulmacası (A translation of the original article: "Fun with genomes: the Mycomuncher DNA Puzzle")
Science in School, 2007: 1(5); 28-31.
A-06: Hikmet Geçkil. Gözler Ufukta, ayaklar yerde: Tim Hunt'la söyleşi (A translation of the original article "Eyes on the horizon, feet on the ground: interview with Tim Hunt") 
Science in School, 2007: 1(6); 9-13.
A-07: Hikmet Geçkil. Romalılara ait yollar, tren istasyonları ve kontrolörler: RNA araştırmalarında son buluşlar (A translation of the original article "Of Roman roads, train yards and inspectors: recent discoveries in RNA research
Science in School, 2007: 1(6); 20-24. 
A-08: Aslı Giray Kurt ve Hikmet Geçkil. Proteini kristallere büyütme (A translation of the original article "Growing crystals from proteins") 
Science in School, 2009: 11; 30-36. 

A-09: Emel Aytan ve Hikmet Geçkil. Folik asit: öğrenciler neden bu konuyu bilmeli? (A translation of the original article "Folic acid: why school students need to know about it") 

Science in School, 2009: 13; 59-64. 

A-10: Zeliha Türkoğlu ve Hikmet Geçkil. Okulda nanoteknoloji (A translation of the original article "Nanotechnology in school") 
Science in School, 2008: 10; 70-75.

A-11: Tuğçe Kaymaz ve Hikmet Geçkil. Mikrobik yakıt hücresi: mayadan elektrik üretimi. (A translation of the original article "The microbial fuel cell: electricity from yeast") 

Science in School, 2010: 14; 32-35.

A-12: Canbolat Gürses ve Hikmet Geçkil. Omurilik hasarı: kök hücreler cevaba sahip mi? (A translation of the original article "Spinal cord injury: do stem cells have the answer?").

Science in School, 2013: 26; 38-43.

A-13: Samet Kocabay ve Hikmet Geçkil. Ölümcül proteinler: prionlar. (A translation of the original article "Deadly proteins: prions").

Science in School, 2010, 15: 50-54.

A-14: Tuğçe Kaymaz ve Hikmet Geçkil. Genetik planımızın ortaya çıkarılması. (A translation of the original article "Laying bare our genetic blueprint"). Science in School, 2013, 26; 20-24.
A-15: Selen Çolak ve Hikmet Geçkil. DNA’da Urasil: bir yanlışlık mı ya da sinyal mi? (A translation of the original article "Uracil in DNA: error or signal?").  Science in School, 2011, 18; 27-31.
A-16: Yasemin Gökçek ve Hikmet Geçkil. Kalem ve kağıtla biyoinformatik:  filogenetik ağaç oluşturma. (A translation of the original article "Bioinformatics with pen and paper: building a phylogenetic tree"). Science in School, 2011, 18; 27-31.
 
 
 

BOOKS CITING OUR WORK

  • Melatonin: Present and Future (by Pedro Montilla López, Pedro Montilla, Isaac Túnez, 2006, Nova Publishers
  • Functional Food Ingredients and Nutraceuticals: Processing and Technologies (by John Shi, Jerry W. King, 2006, Taylor & Francis)
  • Environmental Microbiology: Methods and Protocols. (by John F. T. Spencer, Alicia L. Ragout de Spencer, 2004, Springer)
  • Dictionary of Nutraceuticals and Functional Foods (by Neason Akivah, Michael Eskin, Tamir Snait, Snait Tamir, 2006, CRC Press)
  • Aging and Age-related Diseases (by Michal Karasek, 2006, Nova Science Publishers)
  • Globins and other nitric oxide-reactive proteins (by Robert K. Poole, 2008, Academic Press)
  • Biosystems: Webster's Facts and Phrases: (by Philip M. Parker, 2008, Icon Group International Inc.)
  • Mycorrhiza: State of the Art, Genetics and Molecular Biology, Eco-Function (by Ajit Varma, 2008, Springer-Verlag)
  • Reviews of Environmental Contamination and Toxicology (by David M. Whitacre, 2011, Springer)
  • Breeding for Fruit Quality (by Matthew A. Jenks, 2011, Wiley-Blackwell)
  • Advances in Microbial Physiology (by Robert K. Poole, 2011, Academic Press)
  • Ecoimmunology (by Gregory Demas and Randy Nelson, 2012, Oxford University Press)
  • Advances in Extracellular Space Research and Application (by Ashton Acton, 2011, Scholarly Editions)
  • Coherent Raman Scattering Microscopy (Ji-Xin Cheng et al., 2012, CRC Press)
  • Chitosan-Based Hydrogels: Functions and Applications (Fanglyian Yaoet al., 2012, CRC Press)
  • Dried Fruits: Phytochemicals and Health Effects (by Cesarettin Alasalvar and Fereidoon Shahidi, 2013, Wiley-Blachwell)
  • Fruit and Cereal Bioactives: Sources, Chemistry, and Applications (by Özlem Tokuşoğlu and Clifford A Hall III, 2011, CRC Press)
  • The Know-How of Face Transplantation (by Maria Z. Siemionow, 2011, Springer-Verlag)
  • Fruit Breeding (by Maria Luisa Badenes, 2012, Springer)
  • Bioactives in Fruit: Health Benefits and Functional Foods (by Margot Skinner and Denise Hunter, 2013, Wiley-Blackwell)
  • Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals and Polymers (by Shang-Tian Yang,Hesham El-Ensashy,Nuttha Thongchul, 2013, Wiley)
  • Food Analysis by HPLC (by Leo M. L. Nollet and Fidel Toldra, 2013, CRC Press)
PATENTS CITING OUR WORK
"Bir şeyi büyükanene açıklayamadıkça, o şeyi gerçekten anlamamışsındır"Albert Einstein


Çalışanların Peryodik Cetveli

AKADEMİK DANIŞMANLIK 

  • M.S., Şebnem O. Erenler (2002); Bakteriyel Hemoglobinin Enterobacter aerogenes’de Fizyolojik ve Metabolik Aktiviteler Üzerine Etkisi; İnönü Üniversitesi, Biyoloji.
  • M.S., Nafia C. Ateş (2003); Bakteri Hemoglobin Geni Klonlanmış Enterobacter aerogenes’de Fermantasyon Metabolitlerinin Üretimi ve Solvent Toleransı; İnönü Üniversitesi, Biyoloji Bölümü.
  • Ph.D., Hüseyin Kahraman (2004); Bakteriyel Hemoglobin Geni Klonlanmış Pseudomonas sp.’lerinin Çeşitli Zararlı Aromatik Madde Yıkım Potansiyellerinin Araştırılması; İnönü Üniversitesi, Biyoloji Bölümü.
  • M.S., Salih Gencer(2004); İnönü Üniversitesi, Biyoloji Bölümü Use of the Vitreoscilla hemoglobin for bacterial production of L-asparaginase: en enzyme used in cancer therapy. İnönü Üniversitesi, Biyoloji Bölümü.
  • M.S., Mirac Uckun(2006); Rastgele Mutasyona Tabi Tutulmuş Gram-negatif Bakterilerin Yüksek Düzeyde L-asparaginaz Sentezi İçin Seçilmeleri; İnönü Üniversitesi, Biyoloji Bölümü   
  • M.S., Ufuk Özer (2006); Bakterilerde Dopamin Sentezi; İnönü Üniversitesi, Biyoloji Bölümü
  • Ph.D., Burhan Ateş (2007); (Eş-danışman) Antikanserojenik L-asparaginazın Farklı Gram-negatif Bakterilerde Üretimi, Kimyasal Karakterizasyonu ve Bazı Klinik Uygulamaları; İnönü Üniversitesi, Kimya Bölümü
  • Ph.D., Şebnem O. Erenler (2007); L-Asparaginaz Geninin (ansB) Farklı Gram-Negatif Bakterilere Klonlanması İzolasyonu ve Ekspresyonu; İnönü Üniversitesi, Biyoloji Bölümü
  • M.S., Emel Aytan (2009); Vitreoscilla Hemoglobin Geninin Erwinia herbicola’ya Klonlanması ve L-DOPA Üretimi Üzerine Etkisi; İnönü Üniversitesi, Biyoloji Bölümü 
  • M.S., Salih Gencer (2009); Gastrik Kanserde Matriks Metalloproteinaz (MMP) Genlerinin Ekpresyonları Üzerine Oksidatif Stres ve RNA Müdehalesinin Etkisi; Fatih Üniversitesi, Moleküler Biyoloji ve Genetik Bölümü, İstanbul
  • Ph.D., Aslı Giray Kurt (2012); Vitreoscilla Hemoglobininin Erwinia herbicola’da İkincil Metabolit Akış Dağılımı Üzerine Etkisi; İnönü Üniversitesi, Biyoloji Bölümü  

DEVAM EDEN AKADEMİK DANIŞMANLIK 

  • Ph.D., Veyis Selen (2008 -); Rekombinant Mikroorganizmalar Kullanılarak L-DOPA ve DOPAMİN’in Eşzamanlı Biyosentezi; Fırat Üniversitesi, Kimya Mühendisliği Bölümü  
  • Ph.D., Emel Aytan (2012-); mTOR Sinyal Yolağının Hücre Büyümesi ve Kanserdeki Rolü;İnönü Üniversitesi, Biyoloji Bölümü
  • Ph.D., Özgür Yılmaz (2013 -); Çalışma konusu henüz belirlenmedi; İnönü Üniversitesi, Biyoloji Bölümü

LABORATUARIMIZIN ESKİ ELEMANLARI 

  • Şebnem Özalp Erenler, Yrd. Doç. Dr., İnönü Üniversitesi, Biyoloji Bölümü
  • Burhan Ateş, Doç. Dr., Department of Chemistry, Inonu University
  • Hüseyin Kahraman, Doç. Dr.,  İnönü Üniversitesi, Biyoloji Bölümü
  • Salih Gencer, Postdoktoral araştırmacı, Department of Biochemistry, The Medical University of South Carolina
  • Ufuk Özer, Postdoktoral araştırmacı, Universty of South Carolina 
  • Nafia Canan Ateş, İnönü Üniversitesi, Tıp Fakültesi

Ph.D. Standartları


İMKANLAR

THE GECKIL GENETIC ENGINEERING LABORATORY is located in the west wing of the colossal E-shaped Advanced Research Center (ARC) of Inonu University. The laboratory is fully equipped to support molecular biology and genetic engineering studies. DNA amplification, gene cloning and expression, pathway and metabolic engineering are the type of rearch methodologies being carried out. Some basic tools of the trade in our lab:

  • Apparatus for gel electrophoresis for nucleic acids and protiens, gel imaging system, gel dryers, Southern and Northern blotting
  • PCR: PCR and rt-PCR
  • Chromatographic equipment (e.g., fraction collector) for protein isolation and purification, Western blotting
  • Cell culture room with laminar flow hood, CO2 incubators, inverted microscope
  • Spectrophotometry: UV-Vis spectrophotometers, nanodrop spectrophotometer, UV-Vis transilluminator,  microplate reader
  • Incubators: gyratory and static type, water baths
  • Freezers: refridges (3 –20 oC) and ultralow freezer (–80 oC).
  • Balances: coarse and fine
  • Santrifuges: microfuges, medium and high speed low-temp or ambient-temperature centrifuges
  • Owens: microwave and drying
  • Fermentors (1 liter and 2 liter)
  • Disolved oxygen monitoring system

Some other ARC instrumentation at our disposal:

  • Multi (3) Fluorescence microplate reader 
  • Transmission electron microscopy (TEM)
  • Scanning electron microscopy (SEM)
  • NMR spectrometry
  • Elemental analysisis apparatus
  • Atomic absorption spectrometry (AAS)
  • Infrared spectrometry
  • High-pressure liquid chromatography (HPLC)
  • Gas chromatography (GC)
  • Gas chromatography/Mass spectrometry (GC/MS)
  • X-ray analysis setting
  • X-ray flourescnet spectrophotometry (XRF)
  • Inductively-coupled plasma system (ICP)
  • Liquid nitrogen genertion system


Databases and Computational Tools   

1. Databases

1.1. Sequences

1.1.1. DDBJ 

1.1.2. EMBL

1.1.3. GenBank

1.1.4. GenBank ftp site

1.1.5. TIGR

1.1.6. Wellcome Trust Sanger Institute

1.2. RNA

1.2.1. Rfam: RNA familiy database

1.2.2. RNA base: Databaseof RNA structures

1.2.3. sRNA: Small RNAdatabase

1.3. Comparative & Phylogenetic

1.3.1. COG: Phylogeneticclassification of proteins

1.3.2. TreeBase: Adatabase of phylogenetic knowledge

1.3.3. XREF: Cross-referencing with model organisms

1.3.4. HomoloGene: Genehomologies across species

1.4. SNPs, Mutations & Variations

1.4.1. dbSNP: Singlenucleotide polymorphism database at NCBI 

1.4.2. HapMap: International HapMap Project

1.4.3. HGVbase : HumanGenome Variation Database

1.5. Microarray & Gene Regulation

1.5.1. GEO: Geneexpression omnibus NCBI

1.5.2. Array Express

1.5.3. SMD: StanfordMicroArray Database 

1.5.4. ChipDB

1.5.5. TRRD: Transcription Regulatory region database

1.5.6. TRANSFAC

1.5.7. JASPA

1.5.8. The Signaling Gateway

1.6. Proteins & Interactions

1.6.1. InterPro

1.6.2. ExPASy Proteomics: Expert ProteinAnalysis System

1.6.3. PRIDE

1.6.4. OPD : OpenProteomics Database

1.6.5. BioGrid

1.6.6. BIND

1.6.7. HPRD

1.6.8. DIP

1.6.9. MiMI

1.7. Reaction Pathways

1.7.1. KEGG: Kyoto Encyclopedia of Genes and Genomes

1.7.2. KEGG ftp site

1.7.3. Biocarta

1.7.4. BioCyc

1.8. Enzyme Databases

1.8.1. BRENDA

1.9. Membrane Transporters

1.9.1. TransportClassification Database

1.9.2. TransportDB

1.10. Glycosylation

1.10.1. Functional Glycomics

1.10.2. Bacterial Carbohydrate Structural Database

1.11. Protein Structure

1.11.1. Protein Data Bank

1.11.2. CATH

1.11.3. Protein Information Resource

1.11.4. Structural Classification of Proteins

1.11.5. Swiss-Prot

1.12. Systems Biology

1.12.1. BiGG Database

1.13. Synthetic Biology

1.13.1. Standard Biological Parts

1.14. Other

1.14.1. Gene Ontology

1.14.2. PubMed

 2. Computational Tools

2.1. Genome Browser

2.1.1. NCBI

2.1.2. Ensembl

2.1.3. UCSC

2.2. Sequences Comparison & Alignment:

2.2.1. NCBI BLAST

2.2.2. WU-BLAST

2.2.3. ClustalW: Multiple sequence alignment

2.2.4. CINEMA: Colour interactive editor formultiple alignments

2.2.5. FASTA

2.3. Promotor & Transcription Regulation

2.3.1. Promotor Scan: Promoter regions based on scoring homologies with putative eukaryoticPol II promoter sequences 

2.3.2. MatInspector: Detection of transciption factor binding sites

2.3.3. TESS: Transcription Element Search System

2.3.4. E. coli DNA-Binding Site

2.4. Microarray & Gene Regulation

2.4.1. MIAME Minimum information about amicroarray experiment

2.4.2. MeV: MultiExperiment Viewer

2.4.3. GenePattern

2.4.4. geWorkBench

2.4.5. Bioconductor

2.4.6. Agilent eArray

2.4.7. DAVID

2.5. Membrane Protein Analysis

2.5.1. DAS: Transmembrane sequence prediction

2.6. Proteomics / Mass Spectrometry

2.6.1. ExPASy Tools

2.7. Protein Structure Visualization

2.7.1. PYMOL

2.8. Metabolism

2.8.1. CellNetAnalyzer / FluxAnalyzer

2.8.2. COBRA Toolbox

2.8.3. FBA

2.9. Pathway

2.9.1. GenMapp

2.9.2. KEGG Tools

2.9.3. GSEA: Gene SetEnrichment Analysis

2.9.4. Extreme Pathway Analysis

2.9.5. PUMA2

2.10. RNA folding

2.10.1. Mfold

2.10.2. Sfold

2.10.3. siRNA SelectionProgram

2.10.4. Vienna

2.11. Oligomer Microarray Design

2.11.1. ArrayOligoSelector

2.11.2. OligoArray 2.0

2.11.3. OligoPicker

2.11.4. OligoWiz 2.0

2.11.5. Picky

2.11.6. ROSO

2.11.7. YODA

2.11.8. GoArrays

2.11.9. OligoSpawn

2.11.10. EC Oligos

2.12. Protein 2-D Structure

2.12.1. Chou-Fasman

2.12.2. NNpredict

2.12.3. PredictProtein

2.12.4. SCRATCH

2.13. Protein 3-D Structure

2.13.1. 3D-PSSM

2.13.2. CPHmodels 2.0

2.13.3. UCLA-DOE

2.13.4. Topology of Protein Structure

2.14. Systems Biology

2.14.1. Cytoscape

2.14.2. Systems Biology Workbench

2.14.3. Ingenuity

2.14.4. GeneGo

2.14.5. Gene Designer

2.14.6. Systems Biology Markup Language

2.14.7. Biowww.net

2.14.8. Tools at Weiznmann Institute

LABORATUARDA YAYGIN KULLANILAN PROTOKOLLER

Bilimsel Notasyon

Bilim insanları çoğu zaman çok büyük veya çok küçük yapılarla uğraşırlar. Ör. Bizler gibi biyolojik bilimlerle uğraşan insanlar metrenin milyonda biri olan (mikronmetre) hücreler veya metrenin milyarda biri olan (nanometre) boyutundaki moleküllerle uğraşırken, astronomi ile ilgili bir bilim adamı genellikle milyar yıldız veya milyarlarca ışık yılı uzaktaki bir gök cisminden bahseder. Kısaca, bilim adamları hem çok büyük ve hem de çok küçük rakamlarla uğraşmak zorundadırlar.
 
Bilimsel notasyon, çok büyük veya çok küçük rakamların 10 üssü şeklinde ifadesidir. Böyle bir gösterimde sayı mantis (M) ve 10n olmak üzere iki kısımdan oluşur: M x 10

M (mantis) 1 ila 9 arasında bir tam sayı iken, n herhangi bir sayı olabilir.

Örnek 1: Dünyamızın ekvatordan çevresi= 40,000,000 m

Bilimsel notasyonla gösterim için 4’tten itibaren saydığımızda 7 basamak sayarız. Dolayısı ile 40,000,000 (40 milyon)’un bilimsel notasyonla daha kısa gösterimi 4 x 107 m’dir.

Örnek 2: Bu insan hücresindeki 46 kromozomu yapan DNA’nın hepsine “genom” denir ve yaklaşık 6,000,000,000 baz çiftinden (yani, A=T, GC) oluşur ve bilimsel notasyonla 6x109 baz çifti olarak ifade edilir. 

Böylece, yazılması ve işlem yapılması zor rakamlar daha kolay bir kullanıma kavuşturulur

Bilimsel notasyonda nokta (.) ve virgül (,) işaretinin önemi

Günlük hayatımızda yapmış olduğumuz gösterimlerde nokta ve virgül çoğu zaman biri birinin yerine kullanılır. Ör. Halk için 1,000 TL ile 1.000 TL arasında gösterimde bir fark yoktur ve her ikisi de BİN Türk Lirasını ifade eder.
 
Ancak bilimsel anlamda bu her iki değer biri birinden oldukça farklıdır: 1,000 TL Bin Türk Lirası anlamına gelirken, 1.000 TL Bir Türk Lirası anlamına gelir. Benzer şekilde, örn. 1,554.7 Litre 1554.7 litre anlamına gelir.


Metrik sistem 

Metrik sistem, bilim dünyasınca kabul edilen ölçüm-tartım sistemidir. Burada mil, yard, fit ve inç gibi Amerika Birleşik Devletlerinde kullanılan uzunluk ölçüleri birimleri yerine km, m, cm, mm gibi birimler kullanılırken, galon, ons, libre gibi ağırlık ve hacim birimleri yerine litre, mililitre, kg gibi birimler kullanılır. Bizlerin metrik olmayan sistemi bilmemiz gerekmez. Ancak, metrik sistemin birimlerini ve bu birimlerin birbirine çevrilimini bilmemiz gerekir. Biyolojide ve genel olarak diğer bilimlerde hem büyük ve hem de küçük üniteler kullanılır. Ör. Tipik bir hücre büyüklüğünden bahsederken mikronmetre (µm) ve moleküller arası mesafeden bahsederken nanometre (nm) veya Angström (Å) terimlerinden bahsedilir. Dolayısı ile bu terimlerin kaç cm, mm veya metreye denk geldiğini bilmemiz gerekir.


Mol-Molekül-Avagadro Sayısı

Her elektron, proton, atom veya molekülün 1 molünde sırası ile Avagadro Sayısı (yani ~6.02 x 1023 adet) kadar elkton, proton, atom veya molekül vardır. Diğer bir deyimle, her maddenin 1 molü, o maddenin Avagadro Sayısı kadar parçasından oluşor. Örneğin, 1 mol tuz (NaCl) yaklaşık 56 g olup, Avagadro sayısı kadar NaCl'den veya eğer suda erimiş is 6.02 x 1023 Nave 6.02 x 1023 Cl- iyonu ortaya çıkarır. 


Peki Avagadro Sayısı ne kadar büyüktür? Bunu üç örnekle açıklayalım...

  • Eğer 1 mol atomdaki atomları saniyede 10 milyon hızı ile saysaydık, hepsini saymamız için 2 milyar yıla ihtiyacımız olacaktı. Yani, yukarıdaki örnekteki 56 gram (yani 1 mol)  tuzdaki NaCl moleküllerini saymamız zynı hızla aynı süreyi alacaktı.
  • Karbon atomu (C)'nun Avagadro Sayısı kadarı 1 mol gelir (yani, 12 g). Avagadro Sayısı kadar mol (yani, 6.02 x 1023 mol) C ise dünyamızın (yani yerkürenin) ağırlığı kadar bir ağırlık yapar (yani, ~ 7.2 x 1024 g).
  • Eni 1 mm olan demir paralardan Avagadro Sayısı kadarını dikey olarak arka-arkaya dizer ve bir ucundan diğer ucuna gitmeye çalışırsak ne olur? Şimdi sıkı durun: ışık hızı ile gittiğimizi varsayarsak, paranın bir ucundan diğerine 63,630 yılda varırız (çözüm: 6.02x1023 mm x 1 m/1000 mm x 1 km/1000 m x 1 san/300,000 km x 1 dak/60 san x 1 saat/60 dak x 1 gün/24 saat x 1 yıl/365 gün).

DNA-RNA-Protein

Genetik bilginin DNA'dan RNA'ya ve RNA'dan Proteine akışı moleküler biyolojinin "central dogma" teorisi olarak bilinir. Biomoleküller herhangi bir manipülasyon yani değişikliğe uğratılmadan (örn, boyanmadan) da ışığın belli dalga boylarını soğururlar (absorbsiyon). DNA ve RNA'nın ışığı maksimum absorbe ettikleri dalga boyu 260 nm (nanometre) iken, proteinlerde bu 280 nm'dir. 280 nm'de 1.000 (yani 1) absorbans sulusyonun ml'sinde yaklaşık 1 mg protein olduğunu gösterirken (yani, 1A280= 1 mg protein/ml), DNA için bu 1A260= 50 µg DNA/ml, RNA içinse 1A260= 40 µg RNA/ml'dir. Peki neden, hemen hemen benzer dalga boylarında absorpsiyon yapmalarına rağmen, 20 kat daha düşük bir DNA konsantrasyonu ile proteine eşdeğer bir absorbans elde deriz. Ve de, DNA ve RNA her ikisi de nükleik asit olmalarına rağmen,  daha düşük konsantrasyonda RNA soluyonu (40 ug), DNA'nın daha yüksek olduğu (50 ug) solüsyona eşdeğerde absorbans verir? İpucu: proteinlerde ışığı ilgili dalga boyunda absorbe eden 3 amino asit (aromatik olanlar) varken, DNA ve RNA'da tüm bazlar (A, G, C, T) ışığı ilgili dalga boyunda soğururlar.


Biyolijik hesplamalarda (örn, hücre yüzeyi, hacmi, vb) kullanılan bazı iki ve üç boyutlu şekiller

   Çevresi Alanı
Kare                     4x  x2
Dikdörtgen 2(x+y)  x · y
Üçgen  x + y + z  0.5 (z · h)  (z= üçgenin taban kenarı, h= yükseklik)
Daire  2 · pi · r    pi · r2
Elips pi[1.5(x + y) - (x·y)1/2]   pi · x · y

 

  Yüzey alanı Hacmi
Küp 6x2 x3
Dikdörgenoid 2(x  · y) + 2(x · z) + 2(y · z) x · y · z
Küre 4 · pi · r2 4/3(pi · r3)
Elipsoid belli bir formülü yok 4pi(r · x · y)/3
Silindir 2pi · r · h + 2pi · r2 pi · r2 · h

    
Şimdi, bu formillerin biyolojide nasıl kullanabileceğine dair birkaç örnek verelim:

  1. Hemen hemen küresel olan olan ve çapı 45 µm olan hücrelerden oluşan bir dokumuz var. Bu dokudaki hücrelerin her birinin hacminin kaç µ3 (mikronküp, mikronmetreküp) olduğunu hesplayalım ve 1 cm3dokuda kaç hücre olduğunu bulalım?
  2. Fibroblast hücrelerinin çapı 10 mm olan dairesel cam bir lamel üzerinde kültürü yapılıyor ve hücrelerin jenerasyon süresi (yani iki katına çıkma süresi)  24 saat. Başlangıçta lamel üzerine 10,000 hücre ile başlarsak, ne kadar zamn sonra bu cam plakanın yüzeyi hücrelerle kapatılmış (yani doldurulmuş) olur? (Not: Fibroblast hücrleri  mikroskopta üçgen benzeri yapılarda görünürler.

Hücre çoğalması, büyüme oranı, jenerasyon süresi
Besin sınırlamsı olmadığı zaman hücreler (özellikle bakteriler) üssel (logaritmik) çoğalırlar (yani, 1, 2, 4, 8, 16, 32, 64., vs). Üssel çoğalma:

N(t)= N02t/t0 olarak ifade edilir. Burada Nt zamandaki hücre sayısını; N0 ise t0 zamandaki hücre sayısını ifade eder. veya.
CC0 x 2n  Burada Ct zamandaki hücre sayısını; C0 ise t0 zamandaki hücre sayısını, n ise jenerasyon sayısını ifade eder.
Jenerasyon süresi ise g= t/n
Büyüme oranı ise µ=  1/g

  • Başlangıçta kültür ortamımızda ml'de 2 x 106 hücre bulunsun (yani, 2 x 106 hücre/ml). Bu yoğunluktaki hücre kültürünün 8 saat içinde 1 x 108 hücre/ml'ye ulaşması için kaç jenerasyon (n) geçirmesi gerekir? jenerasyon süresi (g) nedir? ve büyüme oranı (µ) kaçtır?

Yukarıdaki formülden, 1 x 108= 2 x 106  (2n)
2n = 50
nlog2= log50
n= 5.64 (yani, hücreler 5.64 jenerasyon geçirirler, yani 5.64 kez katları şeklinde çoğalırlar)
g= 8/5.64 = 1.42 saat (yani, hücre sayısı iki katına 1.42 saat veya 85 dakikada bir çıkar)
µ= 1/1.42= 0.7/saat (yani, hücrenin büyüme oranı saatte 0.7'dir)

Birim (Boyut) analizi ile problem çözümü

Laboratuvarımızda tercih edilen problem çözme metodudur. Çoğu zaman hesaplamalarımız uzun 4 işlemi gerektiren yapılarda karşımıza çıkar. Boyut analizi yöntemi ile sadece iki işlem (çarpma ve bölme) kullanılarak, hesaplama tek bir düzlem üzerinde yapılır. Birim sadeleştirme yöntemi aynı zamanda hatasız (küsuratlar da hesaba katıldığından) bir hesaplama yapmamızı sağlar. Bu yöntemde şu ana iki durum göz önünde bulundurulur:
 
  1. Verilmiş olan birimler açık şekilde ve yazılıp gerekirse benzer birimleri elde etmek için çevirimler yapılır
  2. Bizden istenen ne ise o sonuca (birime) ulaşmak için tüm sadeleştirmeler yapılır (biraz yaratıcılık gerekir)

Bu tür bir hesaplamayı 4 işlemle oldukça zamanımızı alacak üç örnek üzerinde deneyelim:

  • Elimizde her biri 1 mm eninde olan demir paralardan Avagadro sayısı (6.02 1023 adet) kadarını dikey olarak yan yana koyarsak bir ucundan diğer ucuna ne kadar zamanda (san, dak, saat, gün, yıl) gideriz (bkz. yukarıdaki problem)?
Burada bize verilmiş olan her biri 1 mm eninde olan 6.02 x 1023 adet metal para. Dolayısı ile dikey yan yana konduklarında toplam 6.02 x 1023 mm yapar. Bu değeri en başa bizden isteneni de (ör. yıl olsun) en sona yazarak istenen birimi (yıl) elde etmek için işlemleri bu yönde yaparız:
 

6.02 x 1023 mm   ........................................................................................................   =?yıl

6.02 1023 mm x 1 m/1000 mm x 1 km/1000 m x 1 san/300,000 km x 1 dak/60 san x 1 saat/60 dak x 1 gün/24 saat x 1 yıl/365 gün=  63,630 yıl!!! 
Yani, ışık hızı ile gitsek bir baştan diğerine bu kadar zaman alacaktır.

  • 200 ml 0.1 mM NaOH solüsyonunda kaç mg NaOH bulunur (NaOH, 40 g/mol)?
     

200 ml 0.1 mM NaOH  .........................................= ?mg NaOH
200 ml x 1 L/1000 ml x 0.1 mmol/L x 1 mol/1000 mmol x 40 g/mol x 1000 mg/g= 0.8 mg NaOH

  • 0.01 M 100 ml HCl ile 0.01 M 10 ml NaOH'ı karıştırsanız, yeni solüsyonun pH değeri ne olur? (HCl kuvvetli asit, NaOH is kuvvetli bazdır, Yani, suda % 100 iyonlarına ayrışırlar).
     

0.01 mol/L x 0.1 L= 0.001 mol= 1 mmol HCl
0.01 mol/L x 0.01 L= 0.0001 mol= 0.1 mmol NaOH
fark, 1- 0.1= 0.9 mmol fazla HCl
toplam hacim 110 ml olduğundan, 0.9 mmol/0.110 L= 8.18 mM= 0.00818 M H+      pH= -log [H+]=~ 2.09

Biorad Protein Assay: Bradford

Principle of the assay: The Bradford assay is a colorimetric assay for protein determination that is based on an absorbance shift in the dye coomassie. On the binding of coomassie to protein, the red form of the coomassie dye changes and is stabilized to the blue form of the coomassie dye  – resulting in a shift in absorbance from 465nm to 595nm. As the increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample, this can be used as a measure for the protein concentration of the unknown sample. Unlike some other protein assays, the Bradford assay is quick and easy to perform. However, a note of caution is that elevated levels of detergent will interfere with the assay. Additionally, the assay has a different range of linearity than other protein assays, e.g. 10 times more sensitive than Lowry.
 


Special Reagents:
BSA: Sigma #A2153 (Store as 2μg/ml solution in dH2O)
Bradford Reagent (aka Biorad Reagent): Biorad #500-0006 (Store at 4oC)

Standards: 1 mg/ml BSA stock (dilute 1:10 to get 0.1 mg/ml BSA)

Add       To get  H2O
20 µl  2 µg/ml  780 µl
40 µl  4 µg/ml  760 µl
60 µl  6 µg/ml 740 µl
80 µl 8 µg/ml  720 µl
100 µl 10 µg/ml  700 µl
120 µl  12 µg/ml  680 µl

Protocol:

1) Add 1, 2, 3, or 4 µl of concentrated unknown, and bring volume up to 800 µl with water.
2) Add 200 µl concentrated Biorad reagent and incubate at room temperature for 5 minutes.
3) Assay absorbance at 595 nm.

References
Bradford, M.M. (1976) Analytical Biochem. 40: 15290-15299.
Zor, T. and Selinger, Z (1996) Analytical. Biochem. 236:302-8

Determination of Protein Concentration by the Lowry Assay
Principle of the assay: The Lowry assay is based on the reaction of cupric ions with peptide bonds under alkaline conditions (the Biuret test). Protein samples are mixed with an alkaline solution containing copper sulphate (Cu2+ions) which react with peptide bonds to produce Cu+ ions  – strong reducing agents. Following this reaction, Folin-Ciocalteau reagent is added where upon the Cu+ ions in the solution reactwith the Mo(VI) ions to form molybdenum blue – a complex of Mo(IV) and Mo(V) ions. The blue color of the dye can then be measured at an absorance of 750nm. As the amount of Mo(IV) and Mo(V) complex is dependent on the amount of Cu+ ions which is, itself, dependent on the amount of protein in the unknown sample, the color produced is a direct reflection of protein concentration and, with the use of standards, can allow protein concentration to be determined.The reaction of Cu+ ions with bichronic acid to produce a green color is the basis behind the BCA assay. A disadvantage of both procedures is the need for incubation of the reaction for a length of time (as, for example, compared to the Bradford assay). However, this can be avoided by using the modified Lowry protocol (see Shakir et al., 1994; reference below)
 

Special Reagents
Folin-Ciocalteau reagent:  Sigma #F9252
BSA (for standards):  Sigma #A2153 (Store as 2mg/ml solution in dH2O)

Solutions Required
Solution A: Dissolve 20g sodium carbonate (Na2CO3) and 4g sodium hydroxide in 1 liter of water.
Solution B: Prepare a solution of 2% copper sulphate (CuSO4:5H2O) and  2% sodium potassium tartrate in 100ml water. 
Solution C: Combine 1ml of Solution A with 49ml Solution B.
Solution D: Combine 10 ml Folin-Ciocalteau reagent with 9 ml water.

Procedure
1. Prepare a standard curve of known amounts of BSA and make up to 300μl volume with distilled water. Prepare in duplicate.
2. Dilute an aliquot of unknown samples to 300μl volume with distilled water. Prepare in duplicate.
3. Add 1ml of Solution C to all standards and unknown samples.
4. Mix well and incubate at 37°C for 3 minutes.
5. Remove from water bath and add 100μl Solution D to each standard and sample.
6. Mix well and incubate at 37°C for 3 minutes.
7. Remove 200μl to a microplate and read absorbance at 750nm on the microplate reader.
8. Alternatively, place each sample in a cuvette and read at 750nm in the standard spectrophotometer.

Calculation
1. By comparison of samples to standards, the amount of protein present from each aliquot of unknown solution can be calculated 
2. The concentration of the unknown solution can now be determined by dividing protein amount by the size of the aliquot added. 

 
e.g. 10μl of solution X was diluted to 300μl with distilled water (Sample X) and processed for the assay. After absorbance was measured, sample X was found to contain 10μg protein. As sample X contains 10μl of solution X, the concentration of solution X is therefore 10μg/10μl = 1μg/μl
 

References
1. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193: 265-275.
2. Shakir, F.K., Audilet, D., Drake, A.J. and Shakir, K.M.M. (1994) Analytical Biochem. 216: 232-233.

 
Biuret Protein Assay
The principle of the biuret assay is similar to that of the Lowry, however it involves a single incubation of 20 min. There are very few interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more material. The biuret is a good general protein assay for batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive.

Principle
Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.

Reagent
A formula for biuret reagent is (per liter final volume) 9 gm Sodium potassium tartrate (f.w. 282.22), 3 gm Copper sulfate x 5 H2O (f.w. 249.68), 5 gm Potassium iodide (166.0), all dissolved in order in 400 ml 0.2 M NaOH (f.w. 40.0) before bringing to final volume. The volume can be scaled up or scaled down of course. Discard if a black precipitate forms.

Assay
1.    Volumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
2.    Warm up the spectrophotometer 15 min. before use.
3.    Prepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction coefficient. Using 5 ml color reagent to 1 ml sample a recommended range is 0.5 to 20 mg protein.
4.    Prepare a reference tube with 1 ml buffer.
5.    If possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated.
6.    Use 1 ml sample per assay tube
7.    Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
8.    Read at 550 nm.

Analysis
Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

Comments
The color is stable, but all readings should be taken within 10 min. of each other. As with most assays, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups will give high or low readings, respectively. For Bovine serum albumin we typically obtain a linear relationship between absorbance and amount protein over a range of 0.5 to 20 mg protein. The assay has not been reliable for amounts below 0.5 mg, however the actual sensitive range may extend beyond the upper limit.
 
Phosphate Assay
 
1. Make standards using sodium phosphate at the following uM concentrations: 0, 2, 5, 7, 10, 20, 40, 60, and 80. Use the screw top glass tubes.
 
2. Dry the samples and standards under nitrogen evaporator (Rm 209), and add 150 ul 70% perchloric acid. Oxidize the samples and standards in a heating block (180C, Rm 218) overnight or for 1 hour if the heating block has been pre-heateed.
3. Rinse the sides of the tubes with 830ul xenopure water and vortex.
4. Add 170 ul 2.5% ammonium molybdate (by weight) and vortex.
5. Add 170 ul 10% ascorbic acid (by weight) and vortex. Make the ascorbic acid fresh every time.
6. Incubate for 15 min at 50°C. Allow tubes to cool.
7. Use spectrophotometer to read absorbance at 820nm.
 
DEAE Column
1) Weigh out the appropriate amount of DEAE sephadex and place in a 15 ml conical tube.
2) Wash resin three to four times with zenopure water. Mix gel with water by repeated inversion, then centrifuge at 1,000 - 2,000 rpm; 40C.
3) Wash two times with 100 mM Tris, pH 7.5.
- Equilibrate 5 minutes with gentle inversion prior to spinning down the resin.
4) Equilibrate resin with the starting buffer (sb) for a period of 5 mins.
- Starting buffer is the buffer in which the sample is in. Any salt that is in the sb.
5) Check the pH of the equilibrated mix. It should be approximately 7.56, if not adjust accordingly.
6) Spin gel down again and add sample to the resin.
7) Allow sample to equilibrate with the resin by mixing on the rotary shaker (in cold room) at a speed of 3 for a period of 40 mins.
8) Load gel onto column and collect flow through.
9) Wash column with 5 column volumes of starting buffer.

 
- In actuality, a more rigorous manner in which to check washing is to monitor A280. Once absorbance has leveled off at zero, the column is sufficiently washed.
10) Elute column with the appropriate salt solutions.
- When eluting SMase activity, elute:
a) three times with (1 ml of) 100 mM NaCl.
b) two times with (0.7 ml) of 250 mM NaCl.
 
E. coli Transformation for Subcloning
Properties of E. coli Strains for Subcloning
 
Common laboratory strains of E. coli, like JM109, DH5α™, and XL-1 Blue, are different from their wildtype counterparts. These strains carry some mutations designed to help you propagate plasmids.Typically laboratory strains have a mutation in the recA gene (recA1), a gene involved in recombination. The mutant gene limits recombination of the plasmid with the E. coli genome so that the plasmid inserts are more stable (the recA1 mutation is more effective than the recA13 mutation). Each of these strains also carries the endA1 mutation that inactivates a nuclease that might copurify with plasmids during purification. This mutation helps you to purify higher quality plasmids. Special treatments must be performed on plasmids from strains that do not have this mutation (e.g., RR1, HB101, etc.) to eliminate the nuclease from the plasmid prep (e.g., the Alkaline Protease digestion in the Wizard® Plus SV Miniprep protocol).
 
Common laboratory strains of E. coli are typically defined as K strains or B strains based on the presence of the restriction and modification system that functions around Eco K I or EcoB I, respectively. In a wildtype  K strain, the E. coli will have both the Eco K I restriction enzyme to cleave foreign DNA and EcoK I methylase to protect and mask host DNA recognition sequences. In B strains, the EcoB I restriction enzyme and methylase serve the same purpose. Strains like JM109, DH5α™ and XL-1 Blue are K strains but carry the hsdR17 (rK–, mK+) mutation. This mutation knocks out the EcoK I restriction enzyme but leaves the methylase intact. Therefore, these strains will not degrade plasmid DNA isolated from a B or K strain but will methylate it. This is useful if the DNA must be transferred to a K strain with an intact K restriction and methylation system. 


If you wish to incorporate blue/white selection into your subcloning scheme, you need to transform E. colicarrying a lacZ∆. This mutation deletes a portion of the β-galactosidase gene leaving what is termed the ω-fragment. The plasmid vector supplies this deleted portion, or α-fragment. Once inside the bacterium, the plasmid produces the α-fragment and the E. coli produces the ω-fragment, which combine to make a functional β-galactosidase. If grown on plate containing 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside  (X-gal), the colony will turn blue as a result of  β-galactosidase activity and indicate full complementation of the bacterium by the plasmid. This is termed α-complementation. Blue/White cloning methods use plasmids with a multiple cloning region within the coding sequence of the α-fragment. Disruption of the reading frame due to the presence of the insert will produce a non-functional α-fragment incapable of  α-complementation. These disrupted plasmids are differentiated from the plasmids without insert by the color of the colony (white versus blue), hence the term blue/white selection. Strains like JM109, DH5α™ and XL-1 Blue have the necessary deletion. One difference between these strains lies in how you get the bacterium to produce the ω-fragment. Both JM109 and XL-1 Blue have a second mutation call lacIq. This mutation leads to increased production of the lacI repressor that stops transcription from the lac operon until substrate is present. To relieve this repression, these strains are grown on media containing the non-cleavable lactose analog, isopropyl-β-D-thiogalactopyranoside (IPTG). DH5α™ does not have the lacIq mutation and constantly produces a low level of the ω-fragment through leaky transcription of the lac operon and therefore does not require IPTG for blue/white selection.


E. coli Transformation for Subcloning
 
1. Thaw 50 uL frozen competent cells (AG1, Stratagene #200232) per transformation on ice.
2. Transfer the cells to a pre-chilled Falcon 2059 (round-bottom with cap) tube, and add 0.85 uL 2-mercaptoethanol which is provided with the competent cells.
 
3. Swirl the tube gently for every 2 min while incubating the cells on ice for 10 min.
4. Add an appropriate amount of DNA (1-50 ng) to the competent cells, and incubate on ice for 30 min without disturbing.
5. Heat shock for 45 sec at 42 C and incubate on ice for 2 min.
6. Add 450 uL pre-warmed (42 C) SOC medium (GibcoBRL), and incubate at 37 C with vigorous shaking.
7. Plate the cells on 3 same plates (e.g. 50, 150, 300 uL) with glass spreader.
8. Incubate overnight at 37 C.

 
Screening for Recombinants
The classic method for screening colonies involves performing a plasmid miniprep followed by restriction digestion. Well-isolated colonies are picked from a plate and transferred to culture medium containing the appropriate antibiotic for selection. Proper sterile technique is important. Many different culture media formulations are commonly used for minipreps. Promega recommends LB media supplemented with antibiotics (see page 48) for miniprep cultures to insure that the bacteria do not outgrow the ability of the antibiotic to select for the plasmid. If a rich medium like Terrific Broth is used, the bacteria can grow to very high cell densities and deplete the antibiotic. Once the antibiotic is depleted, the selection pressure to keep the plasmid is removed, and the plasmid may be lost. You can inoculate the colony into 1–10ml of culture medium. If using a high-copy plasmid, 1–5ml (more typically, 1–2ml) is plenty. If you are using a low-copy plasmid, inoculate 10ml. Aerating the culture is very important for maximum cell density. A 17 × 100mm culture tube is fine for 1–2ml. If growing a larger volume, a 50ml sterile, disposable culture tube is better. Incubate the culture overnight (12–16 hours) with shaking (~250rpm). Remember, the greater the surface area, the greater the aeration. You can even grow miniprep cultures in sterile 25–50ml Erlenmeyer flasks. Once the DNA is purified, a portion of the plasmid is screened by restriction digestion. For high-copy plasmids, you can obtain 4–10µg plasmid DNA per purification (1–5ml). For low-copy plasmids, you will obtain 1–3µg plasmid DNA per purification (10ml).  Use 0.5–1µg of plasmid in your digest. Design the digest so that you can easily determine if your plasmid contains insert.

 

Note: Be sure to run uncut plasmid on the same gel for comparison.

Phenol extraction of DNA samples

Phenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.

Protocol
1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.
2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.
3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction.
4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.

5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as discussed below.

DNA extraction, isolation, precipitation, concentration, electrophoresis, etc.

Concentration of DNA by ethanol precipitation
Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20C overnight (1). To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.

Protocol

1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20degC overnight at this stage.
2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes at 4 degC, decant the supernatant, and drain inverted on a paper towel.
3. Add 80% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.
4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry.
5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (termed 10:0.1 TE buffer).
6. It is advisable to aliquot the DNA purified in large scale isolations (i.e. 100 ug or more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable.

Notes on precipitation of nucleic acids
A. General rules
Most nucleic acids may be precipitated by addition of monovalent cations and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70 degC. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4degC. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.

The general procedure for precipitating DNA and RNA is:
1.    Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated,
2.    Add two volumes of cold 95% ethanol,
3. Place at -70degC for at least 30 minutes, or at -20degC overnight. 

or alternatively

1.    Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile water. Mix by inversion and store at -20degC.
2.    Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated.
3. Place at at -70degC for at least 30 minutes or -20degC for two hours to overnight.
* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol.

B. Oligonucleotides
Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol.
Place at -70degC for at least one hour.

C. RNA
Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol.
Precipitate large volumes at -20degC overnight.
Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for five to 10 minutes.

D. Isobutanol concentration of DNA
DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above.

E. Notes on phenol extraction of nucleic acids
The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. Generally, samples are extracted by addition of one-half volume of neutralized (with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a few seconds to form an emulsion. Following centrifugation for a few minutes, the aqueous (top) phase containing the nucleic acid is recovered and transferred to a clean tube. Residual phenol then is removed by extraction with an equal volume of water-saturated diethyl ether. Following centrifugation to separate the phases, the ether (upper) phase is discarded and the nucleic acid is ethanol precipitated as described above.

A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from nucleic acid samples. Following extraction with phenol/chloroform, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above.

Restriction digestion
Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of three standard buffers containing three concentrations of sodium chloride are prepared and used when necessary. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. See the Appendix for a listing of restriction sites present in the M13 (pUC) MCS and a listing of various restriction enzymes, incubation conditions and cut sites.

Protocol
1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube:

    sterile ddH20        q.s (where "q.s." means quantity sufficient)
    10X assay buffer     one-tenth volume
    DNA                  x ul
    restriction enzyme*  y ul (1-10 units per ug DNA)
        Total volume z ul
*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature.
Note: The volume of the reaction depends on the amount and size of the DNA being digested. Larger DNAs should be digested in larger total volumes (between 50-100 ul), as should greater amounts of DNA.

Refer to the vendor's catalog for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer (10X High, 10X Medium, or 10X Low Salt Buffers, or 10X SmaI Buffer for SmaI digestions). Restriction enzymes are purchased from Bethesda Research Laboratories, New England Biolabs, or United States Biochemicals.

2. Gently mix by pipetting and incubate the reaction at the appropriate temperature (typically 37degC) for 1-3 hours.
3. Inactivate the enzyme(s) by heating at 70-100degC for 10 minutes or by phenol extraction (see the vendor's catalog to determine the degree of heat inactivation for a given enzyme). Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis versus non-digested DNA and a size marker, if necessary.

Agarose gel electrophoresis
Agarose gel electrophoresis (2) is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold (made by wrapping clear tape around and extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-forming comb. The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature, depending on the desired separation. When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated. Size markers are co-electrophoresed with DNA samples, when appropriate for fragment size determination. Two size markers are used, phi-X 174 cleaved with restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23 kb. After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.

Protocol
1. Prepare an agarose gel, according to recipes listed below, by combining the agarose (low gel temperature agarose may also be used) and water in a 500 ml Ehrlenmeyer flask, and heating in a microwave for 2-4 minutes until the agarose is dissolved.

                              0.7%               1.0%             2.0%

agarose                1.05 g              1.5 g           3.0 g

20X TAE                7.5 ml              7.5 ml          7.5 ml
ddH2O               142.5 ml           142.5 ml     142.5 ml
EtBr (5 mg/ml)    25 ul                  25 ul           25 ul

total vol               150 ml              150 ml          150 ml

Genetic technology grade (800669) or low gel temperature (800259) agarose from Schwarz/Mann Biotech.
2. Add 20X TAE and ethidium bromide (EtBr), swirl to mix, and pour the gel onto a taped plate with casting combs in place. Allow 20-30 minutes for solidification.
3. Carefully remove the tape and the gel casting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.
4. Add at least one-tenth volume of 10X agarose gel loading dye to each DNA sample, mix, and load into the wells. Electrophorese the gel at 150-200 mA until the required separation has been achieved, usually 0.5-1 hour (100-120 mA for low gel temperature agarose), and cool the gel during electrophoresis with a fan. Visualize the DNA fragments on a long wave UV light box and photograph with a Polaroid camera.

Elution of DNA fragments from agarose
DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol
1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.
2. Melt the slice by incubating the tube at 65degC.
3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.
4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

Kinase end-labeling of DNA
Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-rATP, T4 polynucleotide kinase, and buffer (3). After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.

Protocol
1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:

        sterile ddH2O                q.s 
        10X kinase buffer            1 ul
        DNA                            x ul
        [[gamma]]-[32-P]-rATP        10 uCi
        T4 polynucleotide kinase    1 ul (3U/ul)
                                    10 ul

[[gamma]]-[32-P]-rATP (35020) ICN and T4 polynucleotide kinase (70031) from United States Biochemicals.
2. Incubate at 37degC for 30-60 minutes.
3. Heat the reaction at 65degC for 10 minutes to inactivate the kinase.

Bacterial cell maintenance
Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation (4), XL1BMRF' (Stratagene) for M13 or pUC-based DNA transformation (5), and ED8767 for cosmid DNA transformation (6-8). To maintain their respective F' episomes necessary for M13 viral infection (9), JM101 is streaked onto a M9 minimal media (modified from that given in reference (1) plate and XL1BMRF' is streaked onto an LB plate (1) containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid media are inoculated with a smear of several colonies and incubated at 37degC for 8 hours, and those cultures then are transferred into 50 ml of respective liquid media and further incubated 12-16 hours. Glycerol is added to a final concentration of 20%, and the glycerol stock cultures are distributed in 1.3 ml aliquots and frozen at -70degC until use (1).

Protocol
1. Streak a culture of the bacterial cell strain onto an agar plate of the respective medium, listed below, and incubate at 37degC overnight.

E. coli strain           Agar Medium/Liquid Media
XL1BMRF' (Stratagene)           LB-Tet
 JM101                                         M9
 ED8767                                      LB

2. Pick several colonies into a 12 X 75 mm Falcon tube containing a 2 ml aliquot of the respective liquid media, and incubate for 8-10 hours at 37degC with shaking at 250 rpm.
3. Transfer the 2 ml culture into an Ehrlenmeyer flask containing 50 ml of the respective liquid media and further incubate overnight (12-16 hours) at 37degC with shaking at 250 rpm.
4. Add 12.5 ml of sterile glycerol for a final concentration of 20%, and distribute the culture in 1.3 ml aliquots into 12 X 75 mm Falcon tubes.
5. Store glycerol cell stocks frozen at -70degC until use.

Notes on Restriction/Modification Bacterial Strains:
1.    EcoK (alternate=EcoB)-hsdRMS genes=attack DNA not protected by adenine methylation. (ED8767 is EcoK methylation -). (10)
2.    mcrA (modified cytosine restriction), mcrBC, and mrr=methylation requiring systems that attack DNA only when it IS methylated (Ed8767 is mrr+, so methylated adenines will be restricted. Clone can carry methylation activity.) (10)
3.    In general, it is best to use a strain lacking Mcr and Mrr systems when cloning genomic DNA from an organism with methylcytosine such as mammals, higher plants , and many prokaryotes.(11)
4.    The use of D(mrr-hsd-mcrB) hosts=general methylation tolerance and suitability for clones with N6 methyladenine as well as 5mC (as with bacterial DNAs). (12)
5.    XL1-Blue MRF'=D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F' proAB, lacIqZDM15, Tn10(tetr)].

Fragment purification on Sephacryl S-500 spin columns
DNA fragments larger than a few hundred base pairs can be separated from smaller fragments by chromatography on a size exclusion column such as Sephacryl S-500. To simplify this procedure, the following mini-spin column method has been developed.

1. Thoroughly mix a fresh, new bottle of Sephacryl S-500, distribute in 10 ml portions, and store in screw cap bottles or centrifuge tubes in the cold room.
2. Prior to use, briefly vortex the matrix and without allowing to settle, add 500 ul of this slurry to a mini-spin column (Millipore) which has been inserted into a 1.5 ml microcentrifuge tube.
3. Following centrifugation at 2K RPM in a table top centrifuge, carefully add 200 ul of 100 mM Tris-HCl (pH 8.0) to the top of the Sephacryl matrix and centrifuge for 2 min. at 2K RPM. Repeat this step twice more. Place the Sephacryl matrix-containing spin column in a new microcentrifuge tube.
4. Then, carefully add 40 ul of nebulized cosmid, plasmid or P1 DNA which has been end repaired to the Sephacryl matrix (saving 2 ul for later agarose gel analysis) and centrifuge at 2K RPM for 5 minutes. Remove the column, save the solution containing the eluted, large DNA fragments (fraction 1). Apply 40 ul of 1xTM buffer and recentrifuge for 2 minutes at 2K RPM to obtain fraction 2 and repeat this 1xTM rinse step twice more to obtain fractions 3 and 4.
5. To check the DNA fragment sizes, load 3-5 ul of each eluant fraction onto a 0.7% agarose gel that includes as controls, 1-2 ul of a PhiX174-HaeIII digest and 2 ul of unfractionated, nebulized DNA saved from step 4 above.
6. The fractions containing the nebulized DNA in the desired size ranges (typically fractions 1 and 2) are separately phenol extracted and concentrated by ethanol precipitation prior to the kinase reaction.

Preparation of Genomic DNA from Mammalian Tissue
Tissue is rapidly frozen and crushed to produce readily digestible pieces. The processed tissue is placed in a solution of proteinase K and SDS and incubated until most of the cellular protein is degraded. The digest is deproteinized by successive phenol/chloroform/isoamyl alcohol extractions, recovered by ethanol precipitation, and dried and resuspended in buffer.

Materials
Tissues, whole or cultured cells, Liquid nitrogen Digestion buffer (see recipe) PBS, ice cold 7.5 M ammonium acetate, 70% and 100% ethanol, TE buffer at pH 8, Incubator or water bath at 50°C with shaker.
Additional reagents and equipment for trypsinizing adherent cells and phenol/chloroform/isoamyl alcohol extraction.

Beginning with whole tissue:
1a. As soon as possible after excision, quickly mince tissue and freeze in liquid nitrogen. If working with liver, remove the gallbladder, which contains high levels of degradative enzymes.
2a. Starting with between 200 mg and 1 g, grind tissue with a prechilled mortar and pestle, or crush with a hammer to a fine powder (keep the tissue fragments, if crushing is incomplete).
3a. Suspend the powdered tissue in 1.2 ml digestion buffer per 100 mg tissue. There should be no clumps.

Beginning with tissue culture cells:
1b. Pellet suspension culture out of its serum-containing medium. Trypsinize adherent cells and collect cells from the flask. Centrifuge 5 min at 500 × g, 4°C, and discard supernatant.
2b. Resuspend cells with 1 to 10 ml ice-cold PBS. Centrifuge 5 min at 500 × g and discard supernatant. Repeat this resuspension and centrifugation step.
3b. Resuspend cells in 1 vol digestion buffer. For <3 × 107 cells, use 0.3 ml digestion buffer. For larger numbers of cells use 1 ml digestion buffer/108 cells.

Lyse and digest cells
4. Incubate the samples with shaking at 50°C for 12 to 18 hr in tightly capped tubes. The samples will be viscous (After 12 hr incubation the tissue should be almost indiscernible, a sludge should be apparent from the organ samples, and tissue culture cells should be relatively clear).
Extract nucleic acids
5. Thoroughly extract the samples with an equal volume of phenol/chloroform/isoamyl alcohol. CAUTION: Phenol is extremely caustic.
6. Centrifuge 10 min at 1700 × g in a swinging bucket rotor. If the phases do not resolve well, add another volume of digestion buffer, omitting proteinase
K, and repeat the centrifugation. If there is a thick layer of white material at the interface between the phases, repeat the organic extraction.

Purify DNA
7. Transfer the aqueous (top) layer to a new tube and add 1⁄2 vol of 7.5 M ammonium acetate and 2 vol (of original amount of top layer) of 100% ethanol. The DNA should immediately form a stringy precipitate. Recover DNA by centrifugation at 1700 × g for 2 min. This brief precipitation in the presence of high salt reduces the amount of RNA in the DNA. For long-term storage it is convenient to leave the DNA in the presence of ethanol. Alternatively, to prevent shearing of high-molecular-weight DNA, omit steps 7 to 9 and remove organic solvents and salt from the DNA by at least two dialysis steps against at least 100 vol TE buffer. Because of the high viscosity of the DNA, it is necessary to dialyze for a total of at least 24 hr.
8. Rinse the pellet with 70% ethanol. Decant ethanol and air dry the pellet. It is important to rinse well to remove residual salt and phenol.
9. Resuspend DNA at ∼1 mg/ml in TE buffer until dissolved. Shake gently at room temperature or at 65°C for several hours to facilitate solubilization. Store indefinitely at 4°C. From 1 g mammalian cells, ∼2 mg DNA can be expected. If necessary, residual RNA can be removed at this step by adding 0.1% sodium dodecyl sulfate (SDS) and 1 g/ml DNase-free RNase (UNIT 3.13) and incubating 1 hr at 37°C, followed by organic extraction and ethanol precipitation, as above.

REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. 
Digestion buffer
100 mM NaCl
10 mM Tris⋅Cl, pH 8 
25 mM EDTA, pH 8 
0.5% SDS
0.1 mg/ml proteinase K
Store at room temperature
The proteinase K is labile and must be added fresh with each use.

Southern Blotting
Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support. The transfer or a subsequent treatment results in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified.

Prepare the gel
1. Digest the DNA samples with appropriate restriction enzyme(s), run in an agarose gel with appropriate DNA size markers, stain with ethidium bromide, and photograph with a ruler laid alongside the gel so that band positions can later be identified on the membrane.
2. Rinse the gel in distilled water and place in a clean glass dish containing ∼10 gel volumes of 0.25 M HCl. Shake slowly on a platform shaker for 30 min at room temperature.
3. Pour off the HCl and rinse the gel with distilled water. Add ∼10 vol denaturation solution and shake as before for 20 min. Replace with fresh denaturation solution and shake for a further 20 min.
4. Pour off the denaturation solution and rinse the gel with distilled water. Add ∼10 vol neutralization solution, shake as before for 20 min, then replace with fresh neutralization solution and shake for a further 20 min.

Set up the transfer
5. Place an oblong sponge, slightly larger than the gel, in a glass or plastic dish (if necessary, use two or more sponges placed side by side). Fill the dish with enough 20× SSC to leave the soaked sponge about half-submerged in buffer.
6. Cut three pieces of Whatman 3MM paper to the same size as the sponge. Place these on the sponge and wet them with 20× SSC.
7. Place the gel on the filter paper and squeeze out air bubbles by rolling a glass pipet over the surface.
8. Cut four strips of plastic wrap and place over the edges of the gel.
9. Cut a piece of nylon membrane just large enough to cover the exposed surface of the gel. Pour distilled water ∼0.5 cm deep in a glass dish and wet the membrane by placing it on the surface of the water. Allow the membrane to submerge, then leave for 5 min. If a nitrocellulose membrane is being used, submerge in distilled water; replace the water with 20× SSC and leave for 10 min. Avoid handling nylon and nitrocellulose membranes even with gloved hands—use clean blunt-ended forceps instead.
10. Place the wetted membrane on the surface of the gel. Try to avoid getting air bubbles under the membrane; remove any that appear by carefully rolling a glass pipet over the surface.
11. Flood the surface of the membrane with 20× SSC. Cut five sheets of Whatman 3MM paper to the same size as the membrane and place these on top of the membrane.
12. Cut paper towels to the same size as the membrane and stack these on top of the Whatman 3MM papers to a height of ∼4 cm.
13. Lay a glass plate on top of the structure and place a weight on top to hold everything in place. Leave overnight.

Disassemble the transfer pyramid
14. Remove the paper towels and filter papers and recover the membrane. Mark in pencil the position of the wells on the membrane and ensure that the up-down and back-front orientations are recognizable. Pencil is preferable to pen, as ink marks may wash off the membrane during hybridization.
15. Rinse the membrane in 2× SSC, then place it on a sheet of Whatman 3MM paper and allow to dry.

Immobilize the DNA
16. Wrap the membrane UV-transparent plastic wrap, place DNA-side-down on a UV transilluminator (254-nm wavelength) and irradiate for the time determined from the support protocol.
17. Store membranes dry between sheets of Whatman 3MM paper for several months at room temperature. For long-term storage, place membranes in a desiccator at room temperature or 4°C.

Northern blotting
Principle of the assay: Northern blot refers to a technique developed by James Alwine, David Kemp, and George Stark of Stanford in 1977 to study the  gene expression by detection of RNA/mRNA in samples. Conventionally, this procedure starts with extraction of total RNA or poly A –mRNA by suitable 
method, running gel electrophoresis to separate the RNA based on size, transfer of the RNA to positively charged membrane, labeling of the probe complementary to part of or the entire target sequence, hybridization of the RNA with the probe, membrane washing and detection of signal by x-rays and quantification by densitometry. Alternatively, to cut short the time, pre-made total RNA/mRNA blots are commercially available. The following protocol uses the pre-made total RNA blot for this technique.

Special Reagents:
1) Prime-IT II Random Labeling kit (Stratagene: Catalogue no: 300385).
2) Nick column (GE Health care-Product code: 17-0855-01/17-0855-02).
3)  ExpressHyb Hybridization solution (Clontech, catalogue no: 8015-1/8015-2).
4) Premade total RNA or mRNA blot from a reputed vendor.

Procedure:
Labeling of Probe (Prime-IT II Random Labeling kit):
1) Thaw the radio-nucleotides (3000Ci/mmol-32P dCTP).
2) In a micro centrifuge tube, add the following reagents. 

Ingredients             Control (ul)         Sample (ul)
DNA                         1                           25 ng
ddWater                   23                       0-23
Random primer     10                       10
Total                         34                       34
Mix thoroughly by pipetting.
3) Boil tubes for 5 min. to denature the template DNA; after boiling centrifuge briefly to collect the liquid condensed on the cap;  place the tubes in a 37oC water bath if the DNA sample is in LNT agarose; if it is a gel purified product , place it at  the room temperature before proceeding 
 
to the next step. 
4)  Add the following ingredients into the control and the sample tube as follows

Sl no          Ingredients                                              Control (ul)          Sample (ul)
1                 5x Primer buffer (dCTP)                         10                         10
2                 Labeled nucleotides 3000 Ci/mmol      5                           5
3                 Exo- Klenow enzyme (5U/ul)                   1                           1
4                 Total                                                           16                        16
Mix thoroughly by pipetting.
5) Incubate the reactions for at least 10 min. at 37oC - 40oC.
6) Stop the reaction by adding 2 ul of stop mix, store it in -20oC inside a plexiglass shield and follow the purification protocol outlined below.

Measurement of Probe specific activity:
1) Complete all the 5 steps of the protocol mentioned above.
2) Remove 1ul aliquot at 2 min. intervals and dilute with 99 ul so 0.2M EDTA.
3) Spot 3ul of each time course onto Whatman DE 81 filter paper.
4) Dry 15 min. under heat lamp and place in 5 ml of scintillation fluid and count the cpm. This represents total amount of radioactivity in the reaction mixture.
5) Repeat steps 1-3, this time wash twice for 5 min at  room temperature in 50 ml 2X SSC and once with ice-cold ethanol.
6) Dry the filter paper in the heat lamp again.7) Place in 5 ml of scintillation fluid and count the cpm again. This represents proportion of radionucleotide incorporated into probe DNA.

Specific activity can be calculated from the following formula:
SA = [(μCi)(2.2 x 109)(P)] ÷ {Mi + [(1.3 x 103)(P)(μCi/S)]}
where SA is the specific activity in dpm/μg; μCi is the μCi radiolabeled nucleotide in the reaction mixture; P is the proportion of radiolabeled nucleotide incorporated into probe DNA, calculated by dividing the average cpm counted on the washed DE 81 filter paper disks divided by the average cpm counted on the unwashed DE 81 filter paper disks; Mi is the mass of input DNA template in ng; and S is the specific activity of radiolabeled nucleotide in Ci/mmol (or μCi/nmol).

Purification using Nick column (GE Health care-Product code: 17-0855-01/17-0855-02):
Only reagent needed is TE buffer (10mM Tris and 1 mM EDTA) and manufacturer’s instruction for using the column is given below:
1) Remove the top cap and empty the contents of the upper chamber by decanting.
2) Rinse the column with 3mL of TE buffer and decant it.
3) Place the column on 15 mL plastic tube and pour the column with 3mL of TE buffer and remove the bottom cap. Allow to enter the gel bed by gravity flow.
 
4) Once it is totally emptied, empty the 15 mL tube and place the column back. Apply 100 ul of sample and allow it enter the gel bed. Apply 400 ul of TE buffer and allow it enter the gel bed.
 
5) Place the column in a new 15 mL tube. Add Apply 400 ul of TE buffer and allow it enter the gel bed. Collect the eluate by gravity flow and store the purified sample at  -20oC inside the plexiglass shield until needed.

 
Hybridization Protocol (Adapted from ExpressHyb Hybridization solution user manual with minor modifications):
Notes:
1) Warm the hybridization solution at 68oC in case of precipitation and stir thoroughly to avoid foaming.
2)  Use the following formula to get the hybridization temperature.For probes less than 200 nucleotides in length:
Tm= 81.5+ 16.6 (log10 [Na+]) + 0.41 [{(G+C)/n} (100)]- 600/n
Where ‘n’ refers to the number of nucleotides of the probe and [Na+] refers to the concentration of sodium ions in the hybridization solution, which in this case is 0.5M and G/C refers to number of Guanine and Cytosine bases of the oligonucleotide probe.For probes greater than 200 nucleotides in length:
 
Perform hybridization at 15-25oC less than the Tm as mentioned above.
3) Recommended probe concentration is 20-50 ng/ml or 1-2 x 107 cpm/ml or > 5X 108 cpm/ g.
4) Set the temperature of the water bath to 100oC to denature the probe.
5) Arrange the cassette for the final step.

Procedure:
CAUTION: Follow standard laboratory protocol to handle RNA. 
1)  In a Pyrex dish, shake the blot in 3 cm of DEPC treated water while  preparing for prehybridization. 
2) Warm the hybridization oven to 37oC.
3)  Prehybridize 19.3 cm L x 12.3 cm W membrane in 10 ml of Express hybridization solution in a hybridization bottle with continuous shaking at 37oC for 30 min. 

Step by step instruction of this step:
a) Place 10 mL of hybridization solution in the hybridization bottle. b) Prewarm it by placing inside the oven for 10 min.
c) Roll the blot with RNA side facing in.  Use the mesh to cover that side (Optional- soak the mess in the DEPC treated water overnight and just before the experiment air dry).
d) Use a Tissue culture pipette to roll down gently to make the blot attach to the glass and make sure no air bubbles are there.
e) Put that back into the oven with prewarmed hybridization solution for 20 min.
4) During the 20 min. time prepare the probe. Boil it to 100oC for 10 min and quickly place that back on ice. Quick spin in the radioactive centrifuge. After prewarming the blot for 20 min. take the hybridization bottle and the probe to the radioactivity area. Place a 15 mL tube ready there to mix the probe and hybridization solution. Do not add the probe directly onto the blot. Add radiolabeled probe to 5 ml of fresh Express hybridization solution (10 ml would be better if you have enough probe!). Make sure that you don’t form air bubbles otherwise hybridization would not be perfect. Make sure the solution is covering the entire blot.
 
5) Incubate with continuous shaking at the above calculated temperature  from step 2 of hybridization protocol for 1hr. Make sure you have radioactive dump in the radioactive area (500mL conical flask) so that wash solution can be safely removed.6) Open the oven and reduce the temperature to 25oC (room temp) and keep it open so the temperature is reached quickly. Rinse the blot with wash solution 1 (20 mL of 2x SSC, 0.05% SDS) with continuous shaking at room temperature for 40 min, replace the wash solution several times (4 times for every 10 min interval).
 

7) Wash the blot with wash solution 2 (20 ml of O.1x SSC, 0.1% SDS) two times with continuous shaking at room temperature for 40 min, replace the wash solution  once in between.
8) Remove the blot with forceps and shake off excess solution but do not allow to go dry. It is difficult to remove the probe for re-probing.
7) Immediately cover the blot with plastic wrap.
8) Expose to x-ray film at -70oC with two intensifying screens for sufficient length of time.

Note:
Hybridization temperatures, stringency of washes are some of the factors that should be empirically determined by the researcher to get better results.
Data analysis:  Intensity of band can be quantified by densitometry.

References: 
1) Molecular Cloning: A Laboratory Manual (2001, Third Edition). Joseph Sambrook, Peter MacCallum and David Russell, CSH Protocols, CSH publication.
2) Current Protocols in Molecular Biology (2003). Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl (eds.). John Wiley & Sons Inc.
3) http://www.duke.edu/web/ceramide/protocols/

Mol, Molekül, Molarite, Normalite

Biyokimyasal anlamda molekuller (belli spesifik atomlarin electron paylasimi ile yani kovalent baglarla bir arada tutuldugu yapilar) soludugumuz oksijen (O2) gibi kucuk bir yapidan amino asitlere, nukleotdlere ve hatta protein ve nukleik asitler gibi yapilar icin kullanilir. Mol terimi de ayni yapilar icin kullanilir. Ancak mol molekule gore cok daha buyuk bir niceligi ifade eder ve her maddenin bir molunde sabit sayida molekul bulunur. Buna, Avagadro sayisi denir. Yani her turlu maddenin 1 molunde Avagadro sayisi(6.02 x 1023) kadar molekul vardir (yani, 1 mol glukozda 6.02 x 1023 adet glukoz molekulu, 1 mol H+ iyonunda 6.02 x 1023 adet H+ iyonu, 1 mol e- (elektronda) 6.02 x 1023 adet e- bulunur).

Bu sayede bir elementin veya molekulun gercek agirligi saptanabilir. Ornegin, soludugumuz bir oksijen (O2) molekulunun gercek agirligi 32 g/ 6.02 x 1023 molekul O2= 5.316 x 10-23 g/molekul O2’dir. Bu rakamin kucuklugunu veya Avagadro sabitesinin buyuklugunu tahayyul edebeilemk icin soyle bir ornek dusununuz. Eni 1 mm olan demir paralardan 6.02 x 1023 dikey olarak dizseniz, bir ucundan otekine isik hizi (300,000 km/saniye) ile gitseniz ne kadar surede ulasabilirsiniz? Bunu hesaplayin!!!! Molekullerin ne kadar kucuk veya buyuk degerlerle ifade edilebilecegini diger bir ornekle dusunelim:

Erişkin bir insanda yaklasik 100 trilyon (100 x 1012) cekirdekli hucre oldugu sanilmaktadir ve yine her hucredeki 46 kromozomun toplam 6 milyar (6 x 109) baz cifti (bc)’den olustugu tahmin edilmekte ve iki baz cifti arasindaki mesafenin 0.34 nm (nanometre) oldugu bilinmektedir (1 nm= 1 x 10-9 metre). Yine bilgilerimizden bir baz-ciftinin yaklasik 660 dalton oldugunu (dalton= 1g/6.02 x 1023= 1.66 x 10-24 gram) biliyoruz (kimyacilar tarafindan agirlik birimi olarak kullanilmayan dalton biyokimyacailar tarafindan DNA gibi buyuk molekuller icin pekala kullanilabilir. Neden?). Tum yukaridaki bilgilerden ergin bir insan DNA molekullerinin hepsini uc uca baglarsaniz ceveresi 40,000 km olan dunyayi 5 milyon (5 x 106) kez DNA’mizla sarabilirsiniz. Ancak, bu kadar muazzam uzunluktaki DNA’nin agirligi ise sadece 65 gram kadardir (bunlari gosterebilirmisiniz?).

Biyolojik Bilimler Laboratuvarlarında deney hazirliklarinda en cok kullanilan sozcukler arasinda cozelti (solusyon), konsantrasyon gibi terimler en onde gelir. Bir solusyon iki kisimdan olusur: solut (cozunen) ve solvent (cozen, cozgen). Dolayisi ile seker solut iken, su solventtir ve sekerin suya katilarak eritilmesi ile sekerli solusyon ortaya cikar. Biyokimyada solvent denince en cok su akla gelse de, bircok polar olamayan molekul icin (ornegin yaglar, hidrokarbon bilesikler) yine polar olmayan solventler (ornegin, ethanol, methanol, benzen, toluen, hekzan) cozucu ortam olarak kullanilir. Konsantrasyonlar degisik bicimlerde ifade edilebilir. Bunlar arasinda molar konsantrasyon (M), normal konsantrasyon (N), % konsantrasyonlar (v/v; w/v; w/w) en yaygin kullanilanlaridir.

            Molar konsantrasyon (M) bir litre solusyondaki mol solut sayisidir (M= mol/L, yani, mol solut/L solusyon). Dolayisi ile,

M x L= mole

M x ml= mmol

mM (milimolar) x ml= mmol (mikromol)

mM x ml= nmol (nanomol)

1 L= 1000 ml (mililitre)

1 ml= 1000 ml (mikrolitre)

1 mol = 1000 mmol

1 mmol= 1000 mmol

1 mmol= 1000 nmol

1 nmol= 1 x 10-9 mol

 (ayni iliski M-mM-mM arasinda da vardir)

Bazi ornekler:

Ornek 1. 200 ml 0.5 M NaOH solusyonunda kac mol NaOH vardir?

0.5 mol/L x 0.2 L= 0.1 mol

Ornek 2. 200 ml 0.5 M NaOH solusyonunda kac gram (g) NaOH vardir? (NaOH, 40 g/mol).

0.5 mol/L x 0.2 L x 40 g/mol= 4 g NaOH

Ornek 3. Litresinde 60 g glukoz bulunan solusyonun molar konsantrasyonu nedir? (Glukoz, 180 g/mol).

60 g/L x 1 mol/180 g= 0.333 mol/L= 0.333 M

Ornek 4. Litresinde 60 g glukoz bulunan solusyonun 100 ml’si kac molardir ve kac mol glukoz icerir.

Molar konsantrasyon ayni (yani 0.333 M) kalirken, 100 ml’deki mol glukoz 10 kat azalacaktir (yani 0.0333 mol). 60 g/L x 1 mol/180 g= 0.333 mol/L x 0.1 L= 0.0333 mol, 0.0333mol/0.1 L= 0.333 mol/l= 0.333 M

Ornek 5. 500 ml 1 M glukoz solusyonuna 500 ml su eklerseniz yeni karisimin mol sayisi ve molar konsantrasyonu ne olur?

Mol sayisi ayni (yani 1 mol/L x 0.5 l= 0.5 mol, toplam hacmi 1 L yapsaniz bile yine 0.5 mol madde olacaktir) kalirken, yeni karisimin molaritesi 2 kat dusecektir (1 mol/L x 0.5 l= 0.5 mol, 0.5 mol/2 L= 0.25 mol/l= 0.25 M).

Ornek 6. Konsantre (yogun) HCl (% 35 HCl, d (yogunluk)= 1.15 g/ml, HCl= 36.5 g/mol) solusyonundan 2 litre 0.4 M HCl solusyonu nasil hazirlarsiniz?

Konsantre HCl= 1.15 g/ml x 1000 ml=1150 g (yani 1 litre HCl= 1150 g).

1 mol HCl/36.5 g x 1150 g/L= 31.51 mol/L, ancak HCl % 35 saflikta olduguna gore,

0.35 x 31.51 mol/L= 11.03 mol/L= 11.03 M

Bu  konsantrasyondaki HCl’den 2 litre 0.4 M yeni HCl solusyonu hazirlamak icin,

(2 L x 0.4 M)/ 11.03 M= 0.073 L (yani 73 ml) konsantre HCL alinir uzerine uygun miktarda (1.927 L veya 1927 ml) su eklenerek 2 L’ye tamamlanmis olursa, solusyon 0.4 M olur.

            Normal (N) konsantrasyon solusyonun 1 litresindeki esdeger-mol soluttur (N= esdeger-mol/L). Biyokimyada esdeger mol daha cok ortama verilebilen asit (H+) veya baz (OH-) gruplari ile iliskilidir. Ornegin, HCl icin esdeger sayisi veya tesir degerligi 1 iken, H2SO4 icin 2’dir (HCl’de iyonize olan bir hidrojen bulunurken, H2SO4’de 2’dir, H+ + H+ + SO4-2). Dolayisi ile HCl’in esdeger mol agirligi 36.5 g iken, H2SO4’un esdeger mol agirligi 49 g’dir). Dolayisi ile N=nM olarak tarif edilebilir. n= esdeger sayisi (Yani HCl’in normalitesi molaritesine esitken, H2SO4’un normalitesi molaritesinin 2 katidir). Asagida bazi kimyasallarin toplam oksidasyon sayilari, esdeger gram ve bir molunun gram esdegerleri verilmistir:

Kimyasal              1 mol               Oksidasyon sayisi   Eşdeger-gram agirlik  Gram-esdeger/mol

HCl                           36.5 g              1                                 36.5 g                            1

H2SO4                      98 g                 2                                 49 g                               2

HNO3                        63 g                 1                                 63 g                              1

H3PO4                       98 g                 3                                 32.7 g                          3

Ca(OH)2                    74 g                 2                                 37 g                             2

AlCl3                          133.5 g            3                                 44.5 g                          3

Al2(SO4)3                  342 g               6                                 57 g                             6

 

Ornek 7. HCl, H2SO4 ve H3PO4’un her birinden 1 mol alip toplam hacim 1 litre olacak sekilde bir karisim hazirlarsak, bu karisimin bu maddeler bakimindan normalitesi ne olur?

Karisim bu kimyasalar bakimindan 1 N HCl, 2 N H2SO4 ve  3 N H3PO4 olacaktir.

Ornek 8. 2 litre 3 N NaOH solusyonunda kac esdeger-gram solut (yani NaOH) vardir?

N= esdeger-gram sayisi/ L solusyon

3 esdeger-gram/L=esdeger-gram sayisi/2 L

Esdeger-gram= 6 esdeger-gram NaOH (veya 6 x 40 g/mol= 240 g NaOH/2 L solusyon= 120 g/L x 1 mol/40 g= 3 mol/L= 3 M= 3 N)

Ornek 9. 200 ml’sinde 14 g sulfurik asit olan solusyonun N?

14 g/0.2 L x 1 L x (1 mol esdeger-gram sulfurik asit/49 g)= 1.43 esdeger-gram H2SO4/L

Ornek 10. Iki asit solusyonundan A’nin konsantrasyonu 1.5 N, B’ninki ise 2.1 N’dir. Bu iki asitten kacar ml almaliyiz ki 250 ml 1.65 N solusyon hazirlamis olalim?

A’dan  X ml alirsak, B’den 250-X ml almamiz gerekir. En son hacim ve konsantrasyon 250 ml 1.65 N olacagindan,

(X)(1.5) + (250-X)(2.1)= (250)(1.65)

X=A=187.5 ml, B=62.5 ml

% konsantrasyonlar 100 kisim solusyondaki solut sayisi.

a. Hacim/hacim % konsantrasyon (v/v): 100 ml solusyonda bulunan ml solut.

b. Agirlik/hacim % konsantrasyon (w/v): 100 ml solusyonda bulunan g solut.

c. Agirlik/agirlik % konsantrasyon (w/w): 100 g solusyonda bulunan g solut.

d. % miligram:100 ml solusyonda bulunan mg solut.

Ayrica cok dilut (seyreltik) solusyonlar icin ppm ve ppb terimleri de kullanilir.

ppm (parts per million): milyonda bir kisim anlamina gelir. Orngein, 1 ppm 1mg/L’ye denk gelir.

(yani 1 mg glukozu 1 litre suda cozerseniz bu cozelti veya solusyon glukoz bakimindan 1 ppm olur). ppb ppm’in 1000 kat daha dilue seklidir.

Ornek 11 . % 5’lik (w/v) 500 ml seker solusyonu nasil hazirlarsiniz?

5 g/100 ml solusyon x 500 ml solusyon= 25 g seker (yani, 25 g seker alip bir miktar suda cozerim ve uzerine 500 ml oluncaya kadar su eklerim).

Ornek  12. % 2.5’lik (w/w) 22 g NaCl solusyonunda kac g NaCl vardir?

2.5 g NaCl/ 100 g solusyon x 22g solusyon= 0.55 g NaCl

Ornek   13. % 10’luk (w/w) 200 g NaOH solusyonu nasil hazirlarsiniz?

10 g NaOH/ 100 g solusyon x 200 g solusyon= 20 g NaOH/ 200 g solusyon (diger bir deyimle, 20 g NaOH 180 g solvent (ornegin su veya baska bir solvent)’e ilave edilir. Dolayisi ile burada toplam 200 g solusyonda 20 g NaOH olur. Toplam hacim 200 ml olmaz).

Ornek  14. 200 ml % 10’luk (v/v) etanol solusyonu?

10 ml etanol/ 100 ml solusyon x 200 ml solusyon= 20 ml etanol (20 ml etanol alinir toplam hacim 200 ml olacak sekilde su ilave edilir).

Ornek  15. 0.2 g/ml stok solusyonundan 100 ml 10 mg/ml solusyon nasil hazirlarsaniz?

(100 ml x 10 mg/ml)/ 200 mg/ml= 5 ml stok solusyonu uzerine 95 ml su eklenir.

Ornek  16. % 95’lik (v/v) alkolden, % 70’lik (v/v) 300 ml alkol nasil hazirlarsiniz?

(300 ml x 0.7)/0.95= 221 ml % 95’lik alkol alinip uzerine toplam hacim 300 ml olacak sekilde su eklenir (yani, 79 ml).

Dilusyon: Konsantre (yogun, derisik) solusyonlardan dilut (daha az yogun, seyreltik) solusyonlarin hazirlanmasidir. Dilue edilmis solusyon icin bulunan degerden hareket ederek orijinal solusyonun konsantrasyonunun hesaplanmasi kullanilan dilusyon faktorlerinin kullanilmasi ile mumkundur. Ornegin, 1000 defa dilue olan bir solusyonda madde konsantrasyonu 0.1 mg/ ml ise, orijinal solusyondaki bu maddenin konsantrasyonu 0.1 mg/ml x 1000= 100 mg/ml’dir.

Asit-Baz, pH Kavramı, Tamponlar

Biyolojik molekullerin aktivitelerinin, yapilarinin tam olarak anlasilmasi ancak asid-baz kimyasaini anlamakla olur. Cunku, hucredeki bir cok yapitasi molekul (amino asitler, nukleotidler) ve makromolekul (proteinler, nukleik asitler) asidik veya bazik karakterde olup iyonize olabilirler. Bu molekul ve makromolekullerin tasidiklari elektrik yuk (elektro pozitif veya negatif) onlarin enzimlerle katalize edilen reaksiyonlarda, proteinlerin konformasyonlarinin (ozel yapi kazanma) kararli halde kalmasinda ve diger molekullerle iliskilerinde (ornegin, bazi spesifik proteinlerin DNA’ya baglanmasi) onemli rol oynar. Ayrica, bu molekul ve makromolekullerin saflastirilarak calisilmasi da onlarin iyonizasyon ozelliklerinin bilinmesinden gecer.

Bronsted asid-baz kuramina gore asit hidrojen iyonu (proton) veren bir madde iken, baz hidrojen alan bir maddedir. Dolayisi ile proton veren bir asit ortamda bir baz olusumuna neden olur. Boylece orijinal asit ve yeni olusan baza konjuge asit-konjuge baz cifti denir. Protonu alip asit forma donusen baz baska bir bazdir:

HA                               +                                B-                    «                   A-                                +                                 HB

Konjuge asitA                                    Konjuge bazB                       Konjuge bazA                                              Konjuge asitB

Boylece her iyonizasyon reaksiyonunda iki konjuge asit-konjuge baz cifti vardir. Kuvvetli bir asit veya kuvvetli bir sulu bir ortamda % 100 iyonize olur. Ornegin, HCl solusyonda % 100 H3O+ ve Cl- iyonlarina iyonize olur:

HCl                 +          H2O     «                   H3O+   +          Cl-

Sulu cozeltilerde H3O+ (hidronyum iyonu veya diger bir deyimle suyun konjuge asiti) hidrojen iyonu (proton)’un gercek formudur. Bo nedenle yukaridaki reaksiyon daha basit sekilde,

HCl     «                   H+       +          Cl-

seklinde ifade edilebilir (yani H3O+= H+). Suyun iyonizasyonu iki sekilde ele alinabilir: su basit bir ayrisimla H+ ve OH-‘ya veya  bir konjuge asit-baz cifti olarak. Her iki durumda da suyun amfoterik bir madde oldugu gorulur (yani hem asit ve hem de bazik iyon uretir). Yani, hem proton verebilir ve hem de alabilir. Suyun iyonizasyonu asagida verildigi gibi bir ayrisma (disosasyon) sabitesi (Kd), iyonizasyon sabitesi (Ki) veya su icin spesifik bir sabite ile (Ksu) ifade edilebilir:

Basit Disosasyon                                           Konjuge asit-konjuge baz

HOH   «  H+ +   OH-                                 2HOH   «        H3O+   +             OH-

   Kd= [H+] [OH-]/[HOH]                               Ki=  [H3O+] [OH-]/[HOH]2

Dolayisi ile her mol H+ icin (veya H3O+) 1 mol OH- uretilir. Saf suda, hidrfojen iyon konsantrasyonu ([H+])= 10-7 M oldugundan, hidroksil iyon konsantrasyonu da [OH-]= 10-7 M’dir.

Suyun molaritesi (dsu= 1 g/ml, MAsu= 18 g/mol),

1 g/ml x 1000 ml/litre x 1 mol/18 g= 55.56 M

Dolayisi ile Kd ve Ki,

Kd= (10-7) (10-7)/55.56= 10-14/55.56= 1.8 x 10-16       Ki= (10-7) (10-7)/(55.56)2= 3.24 x 10-18

Ksu= Kd x [H2O] veya Ksu= Ki x [H2O]2 olarak ifade edilebilir.

Bu her iki esitlikten, Ksu= 1 x 10-14= [H+] [OH-] olarak elde edilir. Sulu cozeltilerde dolaysi ile daima [H+] [OH-]= 1 x 10-14  olup, hidrojen iyonu azalirsa, hidroksil iyonu ise ayni oranda artarak bu denge korunmus olur.

            pH hidrojen iyon konsantrasyonunun (veya aktivitesinin) kisa ifadesidir. pH hidrojen iyon konsantrasyonunun negatif logaritmasi olarak tanimlanabilir. Benzer sekilde pOH ise hidroksil iyon konsantrasyonunun negatif logaritmasidir (pH= -log [H+], pOH= -log [OH-]). Sulu solusyonlarda daima Ksu=[H+] [OH-]= 1 x 10-14 oldugundan, pKsu= pH + pOH= 14’tur.

Ornek 1. 0.001 M HCl solusyonunun hidrojen iyon konsantrasyonu, pH, hidroksil iyon konsantrasyonu ve pOH?

HCl kuvvetli bir asit oldugundan % 100 iyonlarina ayrisir: HCl «H+        +          Cl-

Dolayisi ile 0.001 M HCl, 0.001 M H+ ve 0.001 M Cl- iyonlarina ayrisir.

 pH= -log [H+]= -log 10-3= 3

[H+] [OH-]= 1 x 10-14 oldugundan, [OH-]= 1 x 10-14/1 x 10-3= 1 x 10-11 , pOH= -log [OH-]= 11

veya kisaca pH + pOH= 14 oldugundan, pOH= 14-3= 11

Ornek 2. 0.04 M NaOH’in pH?

pOH= -log 4 x10-2= 1.4,                    pH= 14-1.4= 12.6

Ornek 3. 1 x 10-8 M HCl’in pH?

Hepimizin hemen cavap verecegi gibi boyle bir sorunun cevabi pH= 8 degildir. Cunku bir asit ne kadar seyreltik olursa olsun pH’i 7’den buyuk olamaz. Cunku saf suyun pH’i zaten 7’dir ve bir miktar hidrojen iyonu da HCl’den geldiginden, toplam proton miktarinda biraz daha artis olacak ve dolayisi ile pH’da da dusus gorulecektir (hidrojen iyon konsantrasyonu arttikce pH’nin dustugunu hatirlayiniz). Dolayisi ile yukaridaki gibi bir solusyonda X M hidrojen iyonu sudan, 10-8 M hidrojen iyonu da HCl den gelir. Dolayisi ile toplam hidrojen iyonu ([H+])= X + 10-8 M’dir. Hidroksil iyonu ise sadece sudan gelir ve onu da X M olarak ifade edelim.

Biliyoruz ki, [H+] [OH-]= 1 x 10-14 , (X + 10-8) (X)= 1 x 10-14 , X2 + 10-8X- 1 x 10-14= 0

X= (-b ± Öb2-4ac)/2a (negatif degeri hesaba almayin). Geri kalan cozumu yaparak pH’yi hesaplayiniz.

            Canli sistemde bulunan asit ve bazlarin hepsi yukarida bahsettigimiz kuvvetli asit veya kuvvetli bazlar gibi % 100 iyonize olmazlar. Bu cesit asit ve bazlar genellikle kismen (% 10 veya daha az) iyonize olurlar:

HA                  +                     H2O                «                   H3O+ (H+)       +          A-

Konjuge zayif asit         Konjuge baz                          Konjuge asit               Konjuge baz

Bu cesit asit ve bazlara zayif asit veya zayif bazlar denir. pH degisimlerine karsi koyduklarindan, bu cesit maddeler esasen tampon maddeleri olarak kullanilirlar. Dolayisi ile bu maddeler farkli iyonizaston sabitelerine (Ka) sahiptirler.

 

Ka= [H+][A-]/[HA],    [H+]= Ka ([HA]/ [A-]),           her iki tarafin logaritmasini alirsak,

log [H+]= log Ka + log ([HA]/ [A-]), her iki tarafi –1 ila carparsak,

- log [H+]= -log Ka - log ([HA]/ [A-]),           pH= pKa- log [HA]/ [A-], pH= pKa+ log [A-]/ [HA]

bu son formul Henderson-Hasselbalch esitligi olarak bilinir. Bu kullanisli formulle pKa’si ve iyonik konsantrasyonu bilinen bir asit veya bazin pH’si veya pH’si ve iyon konsantrasyonu bilinen asit veya bazin pKa’si hesaplanabilir.

Ornek 4. Biyolojide en yaygin olarak verilen zayif astlerden biri asetik asittir. Bu zayif asidin pKa’si 4.76’dir. Molar konsantrasyonu 0.006 olan asetik asit solusyonunun pH’i nedir?

HA      «        H+       +          A-

0.006-x         x                      x

Ka= [H+][A-]/[HA],    Ka= xx/0.006-x,         % 10 kadari iyonize olan (Ka/0.006) boyle bir asit icin 0.006-x degerindeki x’i ihmal edebiliriz. Boylece,  = x2/0.006,         x=[H+]=

Ornek 5. Ka degeri 1.6 x 10-6 olan zayif bir asidin pH’i, 10-3 M solusyondaki iyonizasyonu, pKa ve pKb degerleri nedir?

HA      «        H+       +          A-

10-3-x             x                    x

Ka=  [H+][A-]/[HA],   (x)(x)/ 10-3-x= 1.6 10-6,           x= [H+]= 4 10-5 M,     pH= 4.398

% iyonizasyonu= [H+]/[HA]= 4 10-5/10-3= 0.04= % 4

pKa, Ka’nin negatif logaritmasi oldugundan, pKa= -logKa= 5.8

pKa + pKb= 14 oldugundan, pKb= 8.2

Ornek 6. Eger 0.16 M 25 ml NaOH solusyonuna 0.34 M 10 ml NaOH eklersek yeni solusyonun molaritesi ne olur?

0.16 mol/l x 0.025 litre = 4 x 10-3 mol=    4 mmol

0.34 mol/l x 0.010 litre = 3.4 x 10-3 mol= 3.4 mmol

Toplam        0.035 litre (35 ml)              = 7.4 mmol

7.4 mmol/35 ml= 0.21 mmol/ml= 0.21 mol/l= 0.21 M

Ornek 7. Yukarida verilen karisimin pH?

pOH= -log [OH-]= 0.68,        pH= 13.32

Ornek 8. Eger 0.16 M 25 ml NaOH solusyonuna 0.34 M 25 ml HCl eklersek yeni solusyonun pH?

0.16 mol/l x 0.025 litre = 4 x 10-3 mol=    4 mmol    NaOH

0.34 mol/l x 0.025 litre = 8.5 x 10-3 mol= 8.5 mmol  HCl

Toplam        0.050 litre (50 ml)              = 4.5 mmol fazla HCl var demektir.

4.5 mmol/50 ml= 0.09 mmol/ml= 0.09 mol/l= 0.09 M HCl

pH= -log [H+]= 1.046

Ornek 9. 200 ml suya kac gram NaOH eklemeliyiz ki solusyonun pH’i 11.5 olsun?

pH=14-pOH,              pOH= 2.5, Hidroksil iyon konsantrasyonu ([OH-]) bulmak icin bu degerin anti logarotmasini (ters logaritmasini) alalim; [OH-]= 3.16 x 10-3 M

3.16 x 10-3 mol/l x 0.2 litre x 40 g/mol= 0.025 g NaOH

Ornek 10. Biyolojide en yaygin olarak verilen zayif bazlardan NH3+’un Ka degeri 1.74 x 10-5’tir. Bu bazin 0.1 M solusyonunun pH?

NH3+ + HOH  «        NH4+   +          OH-

 0.1-x                             x                        x

Ka= [NH4+][ OH-]/[ NH3+],               1.74 x 10-5= x2/0.1                  x2= 1.74 10-6                    x= [ OH-]= 1.32 10-3

pOH= 2.88                 pH=14-2.88= 11.12

Ornek 11. Asetik asit ve sodyum asetatin her birinden 1 mol/l iceren bir solusyonun pH? (Ka= 1.74 x 10-5).

CH3COOH    +   NaOH    __Ka__      NaCH2COO-    +    H+     + H2O

Ka= [NaCH2COO-][ H+]/[CH3COOH]

[ H+]= Ka ([CH3COOH]/ [NaCH2COO-])= 1.74 x 10-5 (1/1)= 1.74 x 10-5, pH= 4.76

veya

pH= pKa + log (tuz/asit)

= 4.76 + log (1/1)= 4.76

Ornek 12. Yukaridaki solusyona 0.1 mol HCl ilave edilirse yeni tamponun pH?

Toplam CH3COOH= 1 + 0.1= 1.1 mol

Toplam NaCH2COO-= 1- 0.1= 0.9 mol

pH= pKa + log (tuz/asit)

= 4.76 + log (1.1/0.9)= 4.67

Ornek 13. 50 ml 0.1 M asetik asite 20 ml 0.2 M NaOH eklenirse yeni karisimin pH?

pH= pKa+ log [A-]/ [HA]

A-= 20 ml x 0.2 M=   4 mmol konjuge baz

HA= 50 ml x 0.1 M= 5 mmol konjuge asit (5 mmol asitin 4 mmolu baza donusur)

Toplam  70 ml         1 mmol fazla asit

pH= pKa + log (baz/asit)= pKa+ log [A-]/ [HA]= 4.76 + log (4/1)= 5.36

Ornek 14. 250 ml 0.2 M asetik asit tamponuna kac gram sodyum asetat ileve etmeliyiz ki pH= 5.0 olsun? (NaCH2COO-= 82 g/mol).

pH= pKa + log [A-]/ [HA],    5.0= 4.76 + log [A-]/ [HA],  0.24= log [A-]/ [HA] logaritmadan kurtarmak icin esitligin ters logaritmasini alirsak, 1.74 = [A-]/ [HA], HA= 0.2 x 250= 50 mmol,

1.74 = [A-]/50,            A-= 87 mmol,             87 mmol/250 ml= 0.348 M,              

0.348 mol/l x 0.250 litre x 82 g/mol= 7.13 g NaCH2COO-

Ornek 15. 0.05 M NaCH2COO- tuz solusyonunun pH?

NaCH2COO-   +    HOH    --------------------   CH3COOH                  +          OH-

0.05-x                                                                        x                                            x

Kh= Ksu/Ka= 1 10-14/1.74 10-5= 5.75 10-10= x2/0.05-x,  (x ihmal edilebilir)

x2= 2.88 10-11, x= [OH-]= 5.36 10-6 M,       pOH= 5.27,               pH= 8.73

Bu orneklerden de anlasilacagi uzere, sulu cozeltilerin pH’lari asagidaki sekillerde ozetlenebilir:

a. saf su icin, HOH ↔ H+   + OH-,   [H+]=[ OH-]= 1 x 10-7 M,       pH=7.0

b. kuvvetli asitler icin, ornegin 10-3 M HCl,              [H+]= 10-3 M,              pH=3.0

c. kuvvetli bazlar icin, ornegin 10-3 M NaOH,          [OH-]= 1 x 10-3 M,      pOH= 3.0,      pH=11.0

d. zayif asit icin,         HA ↔ A-        +          H+     

Ka= [H+][A-]/[HA],  [H+]= Ka ([HA]/ [A-]), buradan bulunan degerin negatif logaritmasi alinarak pH bulunur.

e. zayif baz icin,         B   +  HOH   ↔ OH-  +          BH+,                                   Kb= [BH+][OH-]/[B],                         [OH-]= Kb ([B]/ [BH+), buradan bulunan degerin negatif logaritmasi alinarak pOH bulunur ve 14’tten cikarilarak pH hesaplanir.

f. zayif asit ve onun tuzu veya zayif baz ve onun tuzu,

            pH= pKa + log (baz/asit),      pOH= pKb + log (asit/baz)

g. zayif bir asit ve kuvvetli bazdan olusan tuzun (hidroliz), ornegin asetik asite NaOH ilavesi ile meydana gelen sodyum asetatin hidrolizi, NaCH2COO-   +    HOH--------- CH3COOH     +    OH-

burada CH3COOH     = OH- oldugundan, Kh= [OH-]2/NaCH2COO-,  Kh= Ksu/Ka oldugunu biliyoruz, dolayisi ile Ksu= 1 10-14/1.74 10-5= 5.75 10-10, eger sodyum asetatin konsantrasyonunu biliyorsak Kh= [OH-]2/NaCH2COO- ‘dan hidroksil iyon konsantrasyonunu hesaplar ve onun da negatif logaritmasini alarak pOH ve dolayisi ile pH’yi da hesaplayabiliriz.

h. zayif bir baz ve kuvvetli bir asitten olusan tuzun hidrolizi de aynen yukaridaki gibi bulunur ve pH’i hesaplanir. Sadece, burada pKa yerine pKb degeri kullanilir. Ornegin yukaridaki gibi bir ornegin pKb degeri, pKb= Ksu/Ka.

Ornek 16. 0.2 M NH3 ve 0.3 M NH4Cl iceren solusyonun pH? (NH4 icin Ka= 5.7 x 10-10).

NH4                +          HOH   -----------          NH3                +          H3O+ (H+),      Ka= 5.7 x 10-10

NH3                +          HOH   -----------          NH4                +          OH-,                Kb= Ksu/Ka=1.75 x 10-5

Hidroksil iyon konsantrasyonu ile karsilastirildiginda, hidrojen iyon konsantrasyonu cok kucuk kaldigindan,

Kb= [OH-][NH4]/[NH3],        [OH-]= Kb[NH3]/ [NH4]= 1.75 x 10-5 (0.2)/0.3= 1.17 x 10-5 M

POH= 4.93,    pH= 9.07

Iyonik guc (m), bir tamponun iyonik gucu (aktivitesi veya etkin konsantrasyonu) molar konsantrasyonundan farklilik gosterebilir. Cok seyreltik solusyonlarda bir maddenin aktivitesi konsantrasyonuna esdeger olsa da, yogun solusyonlar icin bu durum soz konusudur ve solutun aktivitesi yogunluguna esit degildir. Bir solusyonun iyonik gucu,

    m= ½ S MZ2  seklinde ifade edilebilir. Burada,

M= ortamdaki iyonlarin gercek molaritesi, Z= iyonun yuku. Ornegin, 0.02 M Fe2(SO4)3 ‘un iyonik kuvveti:

0.02 M Fe2(SO4)3 ® 0.02 M 2Fe+3 + 0.02 M 3SO4-2

0.02 M Fe2(SO4)3 ® 0.04 M Fe+3 + 0.06 M SO4-

iyonik guc= ½ S [(0.04)(3)2 + (0.06)(-2)2] = ½ (0.60)= 0.30 M

bir adet +1 anyon ve bir adet  –1 katyon iceren bir solut icin (ornegin, NaCl, ) m= M olur.

Ornek 17. 25 ml 0.12 M MnCl solusyonu ve 35 ml 0.06 M KCl solusyonunun karisimindaki butun iyonlarin konsantrasyonunu ve bu karisimin iyonik gucunu hesplayiniz.

Mn+2 konsantrasyonu 25 ml x 0.12 M/(25ml+35ml)= 0.05 mol/l= 0.05 M

K+ konsantrasyonu     35 x 0.06 M/(25ml+35ml)= 0.035 mol/l= 0.035 M

Cl- konsantrasyonu     (25 ml x 0.24 M)+( 35 x 0.06 M)/ (25ml+35ml)= 0.135 M

Etkin konsantrasyon (m)= ½ (MMn+2 x Z2 Mn+2   +   MK+ x Z2K+    +    MCl- x Z2Cl-)

                                                               ½ [0.05 x (2)2  +     0.035 x (1)2   +   0.135 x  (1)2]= 0.185 M

Ornek 18. Ka degeri 1.6 x 10-6 olan 1 x 10-3 M zayif bir asitin pH? % iyonizasyonu, pKa?, pKb?

HA       ---------------      H+       +       A-

1 x 10-3-x                      x                    x

Ka= x2/1 x 10-3-x,                   x=[ H+]= 4 x 10-5 M, pH= 4.4

% iyonizasyon= 4 x 10-5/1 x 10-3= 0.04= % 4

pKa- -logKa= 5.8,                  pKb=14-5.8= 8.2

Hucre veya dokularda olusan prosesler belli hidrojen iyon konsantrasyonlari altinda ancak olusabilirler. Canli sistemin hucre ici ve hucre disi pH’si dokudan dokuya farklilik gosterebilir. Hatta bir hucredeki farkli organeller farkli pH’lara sahip olabilirler. Doku, hucre veya organel ancak kendine ozgu pH’larda calisirlar. Dolayisi ile vucut veya hucre pH’sinin hemen hemen sabit tutlmasi gerekir. Olabilecek en kucuk pH degisimleri onemli malfonksiyonlara neden olabilir. Tum canlilar hucrelerinde veya vucut sivilarinda belli bir asit-baz dengesini korumak zorundadirlar. Bu nedenledir ki, biyolojik pH’lar dogal olarak organizmada bulunan tamponlarla belli bir ayarda tutulurlar. Bu dersimizde gorecegimiz gibi, tamponlar oyle maddelerdir ki iyonik ortami eklenen baz veya asit iyonlarina karsi direncli kilarlar. Vucudumuzdaki onemli tampon sisteminden ikisi fosfat ve karbonat tamponlaridir. Karbon dioksit eritrosite girdigi zaman hizlica karbonik asite donusturulur.

Ornek 19. 500 ml 0.01 N HCl’in 0.01 N KOH ile titrasyonunu bir egri yardimi ile gosteriniz (yani eklenen baz (y ekseni) miktarina karsilik gelen pH degerlerini x ekseninde gostererek). Baslangicta hic KOH eklenmedigi zaman yukaridaki konsantrasyondaki asit pH= -log 0.01= 2.00’dir. KOH ilavesinde,

HCl + KOH ® H2O + KCl

Asit ve baz iyonlari esit oluncaya kadar solusyonun pH’i ortamdaki fazla asit iyonlari (titre olmayan) tarafindan belirlenir:

100 ml KOH ilavesinde H+ iyon konsantrasyonu,

  H+=  [HCl]VHCl – [KOH] VKOH / (VHCl + VKOH) =  0.004 M   pH= 2.18

benzer sekilde,

200 ml KOH ilavesinde pH= 2.39

300 ml KOH ilavesinde pH= 2.60

400 ml KOH ilavesinde pH= 2.95

Esdeger miktar ve konsantrasyonda baz ilevesi halinde ise, yani,

500 ml KOH ilavesinde pH= 7.00 ([H+] x [OH] = 10-14, burada [H+] = [OH]’dir) olur (cunku ikisi de % 100 iyonize olabilen monobazik ve monoasidik turler oldugundan, ortamdaki bazik ve asidik iyon konsantrasyonlari esit olur. Yani solusyonun pH’i notr olur).

KOH eklemeye devam edersek, titrasyonun ucuncu kisminda hidroksil iyon konsantrasyonu hidrojen iyon konsantrasyonunu gececektir. Dolayisi ile,

600 ml KOH ilavesinde OH- konsantrasyonu,

OH-=  [KOH] VKOH – [HCl]VHCl / (VKOH + VHCl) = 0.00091M

pOH= 3.04, pH= 10.96

700 ml KOH ilavesinde pH= 11.22

800 ml KOH ilavesinde pH= 11.36

900 ml KOH ilavesinde pH= 11.46

1000 ml KOH ilavesinde pH=11.52

1500 ml KOH ilavesinde pH= 11.70

Bu konsantrasyonda (0.01 M) ne kadar KOH eklersek ekleyelim solusyonun pH’i 12’yi gecmez. Cunku, bu bazin kendisinin (asit olmadan) pH’i 12.00’dir. pH 12 veya daha yuksek pH’lar elde temek icin yuksek konsantrasyonda KOH kullanmak gerekir.

Titrasyon egrileri bir solusyondaki asit (veya baz) ‘in miktarini ve o asitin pKa’sini bulmaya yarar. Belli hacimdeki asite belli konsantrasyondaki bir baz (genellikle (NaOH) notralize oluncaya kadar ilave edilir Asitin konsantrasyonu ve pKa’si harcanan ve konsantrasyonu bilinen bazin yardimi ile hesaplanir (bu konuya laboratuvarimizda detayli giris yapilacaktir). Yukaridaki ornegin titrasyon egrisi asagidaki gibi gosterilebilir:

20. Simdi de 500 ml 0.1 M zayif asidin 0.1 M KOH ile titrasyon egrisini saptamaya calisalim (pKa= 5.0).

Yukaridaki orneklerden de anlasilacagi uzere, bir zayif aside kuvvetli baz ilavesi 4 ana basam icinde irdelenebilir ve pH hesaplanabilir:

A. Baslangic bolgesi (henuz kuvvetli baz ilavesi olmadan):

  HA       ---------       H+   +   A-                     Ka= x2/0.01-x,            x= [H+], pH= 3.0

0.1-x                         x            x

B. Ayni esdeger mol kuvvetli baz ilavesi noktasina gelineye kadar:

Herndersson-Hasselbalch esitligi ile pH hesaplanir.

Ornegin, 100 ml KOH ilavesi halinde pH,  0.1 mol/l x 0.1 litre= 0.01 mol OH- ilavesi yapilmis demektir, boylece, zayif asitin 0.01 molu notralize olacaktir ve ortamda, 0.1 mol asit/l x 0.5 litre- 0.01 mol notralize olacak= 0.04 mol asit kalackatir. Dolayisi ile pH,

pH= 5 + log (0.01/0.04)= 4.4

C. Esit molar konsantrasyonda kuvvetli bazin ekendigi bolge (Denge noktasi):

Eger 500 ml 0.1 M KOH ilave edilirse, teorik olarak asitin hepsi notralize edilmis olacaktir. Yani teorik olarak tum HA A-‘ya donusmus olur. Ancak bu noktada, kuvvetli asit ve baz titrasyonunda oldugu gibi pH 7.0 degildir. Cunku, bir kisim tuz (A-) veya konjuge baz iyonize olur.

A-                    +          HOH   «   HA   +   OH-

0.05-x                                            x             x

bu nedenle 1 mol KOH ilave etmek 1 mol A- aciga cikarmaz,

A-= 500 x 0.1/ 500 + 500= 0.05 M

Kh= Ksu/Ka= 10-14/1 x 10-5= 1 x 10-9,  Kh ayni zamanda= [HA][OH-]/[A-]= x2/0.05-x

1 x 10-9= x2/0.05,                    x= [OH-]= 1.58 x 10-6,                       pOH= 5.15,               pH=8.85

D. Denge noktasindan sonraki pH:

Burada eklenmis olan KOH molar konsantrasyonu zayif asitinkini gecer. Ornegin, 600 ml KOH ilavesi halinde, 600 ml x 0.1 M= 60 mmol OH-

                                      500 ml x 0.1 M= 50 mmol HA

Dolayisi ile daha fazla (10 mmol) hidroksil iyonu bulunur.  Toplam hacim 1100 ml oldugundan,

[OH-]= 10 mmol/ 1100 ml= 9.091 x 10-3 M,  pOH= 2.04,               pH= 11.96

Biyolojik sistemler laboratuvar ortaminda daha cok parcalanarak calisiliralar. Ancak, hucre icindekine benzer (suni) bir ortamin yapilamasi gerekir ki calistigimiz madde aktivitesini kaybetmesin. Bu tur calismalara in vitro (ornegin tup icinde olan) calismalar denir. Eger, calismamizda direkt bir canliyi oldurmeden kullaniyorsak buna da in vivo calisma denir. Biyokimyasal reaksiyonlar ortam pH’sina bagimli olduklarindan, hidrojen iyon konsantrasyonunun dogru olarak belirlenmesi bu tur calismalarda her zaman onemli yer tutar.

            Butun amino asitler iyonize olabilen gruplara (amino ve karboksil) sahip olduklarindan, ortam pH’sina bagli olarak asit veya bazik karaktere sahip olabilirler. Bildiginiz gibi butun amino asitler

genel formulune sahiptirler. Glisin en basit amino asit olup R’in pozisyonunda ikinci bir hidrojen atomu tasir. Dolayisi ile en basit amino asitte bile iki adet pKa degeri vardir. Birincisi, karboksil grubunun iyonizasyonuna denk gelen asidik pKa1, ikincisi ise amino grubunun iyonizasyonunda olan bazik tabiyatli pKa2 (Glisin icin pKa1= 2.4, pKa2= 9.6’dir). Dolayisi ile butun amino asitler icin bu pKa degerleri benzer (ancak ayni olmayan) degerler gosteririr. Ancak, bazi amino asitler (ornegin, asidik ve bazik amino asitler) birden fazla amino veya karboksil grubu tasidiklarindan, ikiden fazla pKa degerlerine sahiptirler. Anacak, bilmekteyiz ki peptid bagi yapimina girmeyen bu gruplar proteine belli bir iyonik karakter gosterme ozelligindedirler. Dolayisi ile glisin, valin, losin, izolosin gibi amino asitleri yuksek oranlarda icren proteinler (ornegin: kollojen, keratin) zayif iyonizasyon gosterirler. Neden? Amino asitlerin asit-baz karakteristikleri, pKa’lari, pI’lari ve tampon ozellikleri uzerinde laboratuvarda daha detayli durulacaktir.


PROJELER

  • 2000-2001: Geçkil, H. ve Erenler, Ş.O., “Bakteri hemoglobin genininEnterobacter aerogenes’in fizyolojik ve metabolik aktiviteleri üzerine etkisi”. Lisans Üstü Araştırma Projesi, Proje no: 1999/08  (İnönü Üniversitesi), Proje Yöneticisi, Proje bütçesi: 803.250.000 TL
  • 2001-2002: Erenler, Ş.O., Yılmaz, H.R., ve Geçkil, H., “Solventlerin hemoglobin geni klonlanmış Enterobacter aerogenes’in metabolik ve fizyolojik durumuna etkisi”. Kapsamlı araştırma projesi, Proje no: APYB 2001/16 (İnönü Üniversitesi), Proje Yöneticisi, Proje bütçesi: 2.450.000.000 TL
     
  • 2002-2003: Geçkil, H., Kahraman, H., Erenler, Ş.O., ve Ateş, N.C., “Bakteri hemoglobin geni klonlanmış Pseudomonas aeruginosa’nın endüstriyel kullanım potansiyeli”. Lisans Üstü Araştırma Projesi, Proje no: APYB 2002/05 (İnönü Üniversitesi), Proje Yöneticisi, Proje bütçesi: 6.000.000.000 TL
     
  • 2002-2003: Yürekli, F., Yiğit, E., Geçkil, H. ve Porgalı, Z.B., “Bakır (Cu) ve kadmiyum (Cd) uygulamasına bağlı olarak Pisum sativum L. (Bezelye)’da fitokelatin düzeyleri ve detoksifikasyondaki rollerinin araştırılması. Kapsamlı araştırma projesi”, Proje no: 2002/14 (İnönü Üniversitesi), Yardımcı araştırmacı, Proje bütçesi: 5.230.000.000 TL
     
  • 2002-2004: Geçkil, H., Gencer, S., Erenler, Ş.O., Kahraman, H. ve Ateş N.C., “Vitreoscilla hemoglobin geni klonlanmış Enterobacter aerogenes: bazı endüstriyel uygulamalar”, TÜBİTAK Projesi, Proje no:TBAG2267(102T197),  Proje Yöneticisi, Proje bütçesi: 64.000.000.000 TL
     
  • 2002-2004: Geçkil, H., Yeşilada, E, Yeşilada, Ö., ve Özmen, M., “Biyoteknolojik yöntemlerle tekstil boyalarının yıkımı: biyoteknolojik işlem görmüş ve görmemiş boyar maddelerin toksik/genotoksik etkilerinin farklı test sistemleri kullanılarak saptanması”, Kollektif DPT Projesi, Proje no: (2003K 120610), Proje Eş-Yöneticisi,Proje bütçesi: 220.000.000.000 TL
     
  • 2004-2005: Yılmaz, İ., Geçkil, H., ve Ateş, B., “Bakteriyel orijinli antneoplastik bir enzim olan L-asparaginazın üretimi, kimyasal karakterizasyonu ve klinik farmakolojisi”. Proje no: APYB 2004/93 (İnönü Üniversitesi),Proje yöneticisi, Proje bütçesi: 8.000.000.000 TL
     
  • 2004-2005: Geçkil, H., Aydın, S., Erenler, Ş. O., Gencer, S., ve Uçkun, M., “L-asparaginaz geninin (ansB) farklı Gram-negatif bakterilere klonlanması, izolasyonu ve L-asparaginaz üretimi:kanser kemoterapisinde kullanılan önemli bir enzim”, DPT projesi (2005 Yılı yatırım cetveli, Teknolojik araştırma sektörü). Proje yöneticisi, Proje no: DPT 2003/K120610, Proje bütçesi: 70.000.000.000 TL.
  • 2005-2006: Hikmet Geçkil ve Şebnem Özalp Erenler, “L-asparaginaz geninin (ansB) farklı Gram-negatif bakterilere klonlanması, izolasyonu ve L-asparaginaz üretimi: kanser kemoterapisinde kullanılan önemli bir enzim”.Proje yöneticisi, Proje no: APYB 2005/25 (İnönü Üniversitesi), Proje bütçesi: 9000 YTL. 
  • 2004-2005: Hikmet Geçkil, Burhan Ateş ve Şebnem Özalp Erenler, “Bakteriyel orijinli L-asparaginazın üretimi, kimyasal karakterizasyonu ve klinik yönleri”.Proje yöneticisi, Proje no: APYB 2004/93 (İnönü Üniversitesi), Proje bütçesi: 9000 YTL.
  • 2006-2008: Birol Mutlu, Hikmet Geçkil ve Şükrü Karakuş, “Türkiye’dekiErysimum genusu üzerine taksonomik çalışmalar”. Araştırmacı, TÜBİTAKProjesi (TBAG 105T126), Proje bütçesi: 95,065 YTL.
          
  • 2007- 2009: Hikmet Geçkil, Burhan Ateş, Şule Bulut ve Aslı Giray Kurt, “Vitreoscilla hemoglobini ekspresyonu yapan bakterilerde L-DOPA ve dopamin üretimi”. Proje yöneticisi,TÜBİTAK projesi (TBAG 107T478), Proje bütçesi: 136,000 YTL ve 1500 YTL/ay 2 yıl doktora bursu.

PROJE HAKEMLİK VE DANIŞMANLIK 

2005 yılından beri TÜBİTAK:

  • TEMEL BİLİMLER ARAŞTIRMA GRUBU – TBAG
  • SAĞLIK BİLİMLERİ ARAŞTIRMA GRUBU – SBAG
  • KAMU ARAŞTIRMALARI GRUBU – KAMAG
  • SOSYAL VE BEŞERİ BİLİMLER ARAŞTIRMA GRUBU – SOBAG

             herhangi birinde düzenli olarak proje panelisti

2008 yılından beri TÜBİTAK:

  • TEKNOLOJİ VE YENİLİK DESTEK PROGRAMLARI BAŞKANLIĞI (TEYDEB) AGY100 düzenli firma hakemliği
  • 2007: TÜBİTAK Bilim İnsanı Destekleme Daire Başkanlığı tarafından desteklenen Eğiticilerin Eğitimi Çalıştayı
  • 2008-2009 TÜBİTAK destekli Doğu Anadolu bölgesine yönelik üstün yetenekli “Orta Öğretim Öğrencileri Bilim Yaz Kampı” ve “Araştırma Projeleri Yarışması”nda Biyoloji 19-26 Haziran 2009 İnönü Üniversitesi Malatya
  •  2008-2010: TÜBİTAK Bilim Olimpiyatları (Biyoloji) Eğitmeni
  • 2012: TÜBİTAK tarafından aday gösterilerek OIC (Organisation of Islamic Cooperation) bünyesindeki COMSTECH (Committee on Scientific and Technological Cooperation)'da "Endüstriyel Biyoteknoloji" alanında Türkiye'yi temsil. 
  • 2010-2012: İnönü Ünicersitesi, Üstün Yeteneliler Araştırma Merkezi Kurucu Müdürü
  • 2010-2012: İnönü Ünicersitesi, İnönü Çocuk Üniversitesi Kurucu Koordinatörü

 

 

 

 

 

(figure from Geckil et al. Nanomedicine, 2010; 5(3): 469-484)