BY1101 Introduction to Molecular and Cellular Biology

1. BY1101 Introduction to Molecular and Cellular Biology Tutorial for module BY1101: Chromatography Joe Colgan (tcolgan@tcd.ie)

2. BY1101 Introduction to Molecular and Cellular Biology Tutorial objectives • Describe chromatography • Describe the different types and why they were used in the BY1101 practicals

3. BY1101 Introduction to Molecular and Cellular Biology Column chromatography Column chromatography • What is chromatography? • A set of lab techniques to separate mixtures • Mobile phase: • Fluid that houses the mixture to be separated • (e.g. Cell lysate, haemoglobin, mould extract) • Stationary phase: • Structure holding another material that interacts with and aids in separation of mixture • (e.g. Sepharose, sephadex, DEAE-cellulose) Mobile Stationary

4. BY1101 Introduction to Molecular and Cellular Biology Three types of column chromatography used in the BY1101 practicals. What are they? Practical 2 Practical 3 Experiment 2 Practical 3 Experiment 1 Affinity chromatography Gel filtration chromatography Ion exchange chromatography Separate molecules based on size Separates molecules based on charge Separates molecules based on biological specificity

5. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography What do we use affinity chromatography for? • Purpose • Purification and concentration of biomolecules, such as proteins, on the basis of their biological specificity • Applications • Purification of antibodies • Purification of enzymes

6. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography What was the purpose of Practical 2 – Experiment One? • Glutathione S-transferase (GST): • Catalyzes conjugations of the substrate glutathione (GSH) • GST is an enzyme and binds GSH in an enzyme-substrate complex • Enzyme (E) + Substrate (S)  Enzyme-substrate complex • Interaction is dynamic and GST will bind reversibly Substrate Enzyme Enzyme-substrate complex GST GSH

7. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography What is thesource of the GST? Natural source Recombinant protein

8. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography What is a recombinant protein? A protein encoded for by a gene – recombinant DNA – that has been cloned into a system that supports its transcription and translation l P O gstgene Transcription mRNA Translation l= lacoperon P= Promoter O= Operator Repressor protein

9. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography How do we get “overexpression” of a protein? Isopropyl-beta-D-thio-galactoside (IPTG): IPTG binds to the repressor protein and inactivates it l P O gstgene mRNA Transcription of mRNA l= lacoperon P= Promoter O= Operator IPTG Repressor protein Within the present experiment, what would be the purpose of adding IPTG to the bacterial culture?

10. BY1101 Introduction to Molecular and Cellular Biology Molecule of interest Mobile phase Stationary phase Escherichia coli (bacterial) lysate Sepharose beads coated with glutathione Glutathione S-transferase Are the proteins present in the cell lysate in their native (active) state or are they denatured? Would you expect the proteins present in the lysate to exhibit their natural biological activity? Why was it important to keep the cell lysate on ice?

11. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography Sepharose Glutathione (GSH) Glutathione S-transferase (GST) Cell lysate Sepharose-Glutathione Stationary phase Mobile phase

12. BY1101 Introduction to Molecular and Cellular Biology • Pour the column • (Sepharose Beads-Stationary phase) • Wash the column • (Phosphate buffered saline) Mobile Stationary

13. BY1101 Introduction to Molecular and Cellular Biology 3. Run the column (E. coli lysate) Contains enzyme of interest - GST within lysate binds to glutathione-sepharose beads complex 4.(PBS) Washes the column - Other bacterial proteins are washed out of the column leaving only GST bound to sepharose beads Would you expect GST to be in fraction one? Mobile Stationary Why does GST remain in the column after the PBS washes? 1 2 3 4 5 6

14. BY1101 Introduction to Molecular and Cellular Biology 3. Run the column (E. coli lysate) Contains enzyme of interest - GST within lysate binds to glutathione-sepharose beads complex 4. (PBS) Washes the column - Other bacterial proteins are washed out of the column leaving only GST bound to sepharose beads 5. (Glutathione) Substrate of enzyme - High concentration of glutathione displaces GST from the beads, binds to GST and is eluted out of the column Mobile Stationary 1 2 3 4 5 6 7

15. BY1101 Introduction to Molecular and Cellular Biology Affinity chromatography GST binds to GSH-Sepharose beads Free GSH bind GST and elutes Non-bound proteins removed Column is washed Addition of free GSH 1 7 All proteins in lysate except GST PurifiedGST

16. BY1101 Introduction to Molecular and Cellular Biology SDS-PAGE preparation • Precipitation of protein with trichloroacetic acid (TCA) • Pellet the precipitated protein by centrifugation • Dissolve precipitated protein in sodium-dodecyl sulphate (SDS) • Boil the protein samples

17. BY1101 Introduction to Molecular and Cellular Biology Gel filtration chromatography Practical 3- Experiment One • Purpose • Separation of macromolecules based on size • Applications • Determination of relative molecular size • Separation of molecules on the basis of size • Removal of inorganic ions from preparation of protein

18. BY1101 Introduction to Molecular and Cellular Biology Gel filtration chromatography Direction of flow

19. BY1101 Introduction to Molecular and Cellular Biology Gel filtration chromatography Practical 3- Molecule of interest Oxyhaemoglobin Haemoglobin Purple/red colourOxygen-depleted blood Venous blood Scarlet/red colourOxygen-rich blood Arterial blood

20. BY1101 Introduction to Molecular and Cellular Biology Gel filtration chromatography Practical 3- Molecule of interest Haemoglobin Methaemoglobin Haemoglobin Oxidation Reduction Oxidation: Potassium ferricyanide + Haemoglobin  Oxidised haemoglobin (methaemoglobin) Reduction: Ferrous sulphate + methaemoglobin  Reduced haemoglobin

21. BY1101 Introduction to Molecular and Cellular Biology Mobile • Pour the column • (G-25 Sephadex beads-Stationary phase) • Wash the column • (20mM PBS, pH 7) Stationary

22. BY1101 Introduction to Molecular and Cellular Biology • Add the reducing agent • (40mM FESO4+ 80mM Na2EDTA) • 4. Add methaemoglobin Mobile Stationary Methaemoglobin Reducing agent Haemoglobin

23. BY1101 Introduction to Molecular and Cellular Biology Gel filtration chromatography Addition of reducing agent Addition of methaemoglobin Reduction of methaemoglobin to haemoglobin

24. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography • Purpose • Separation of molecules on the basis of charge • Applications • Water softening, purification and decontamination

25. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Separation of molecules on the basis of charge Direction of flow

26. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Molecules of interest Mobile phase Stationary phase Aspergillusniger (fungal) extract DEAE- Cellulose Glucose oxidase Catalase

27. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography • Stationary phase: • Diethylaminoethyl (DEAE) cellulose • Positively charged (protonated) • Interacts with negatively charged molecules (anions) • Anion exchanger: Stationary phase is positively charged

28. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Separation of molecules on the basis of charge • Glucose oxidase: • Oxidation of glucose to hydrogen peroxide and glucono-1,5-lactone, which hydrolyzes to gluconic acid • Glucose oxidase (GO) requires cofactor flavin adenine dinucleotide (FAD) Glucose + GO:FAD  Glucono-1,5-lactone + GO:FADH2 GO:FADH2 + O2  GO:FAD + H2O2 Glucose oxidase Substrate Enzyme Enzyme-substrate complex Glucose oxidase Glucose

29. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Separation of molecules on the basis of charge • Catalase: • Catalyzes the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2) 2H2O2 2H2O + O2 Catalase Substrate Enzyme Enzyme-substrate complex Catalase Hydrogen peroxide

30. BY1101 Introduction to Molecular and Cellular Biology • Pour the column • (DEAE-Cellulose-Stationary phase) • Wash the column • (PBS) Mobile Stationary

31. BY1101 Introduction to Molecular and Cellular Biology 3. Run the column (Mould extract-Aspergillusniger) 4. Wash the column (Buffer 1- 20mM NaOAc, 5mM acetic acid) Mobile Stationary How do we get our enzymes of interest out of the column?

32. BY1101 Introduction to Molecular and Cellular Biology 3. Run the column (Mould extract-Aspergillusniger) 4. Release the bound molecules (Buffer 1- 20mM NaOAc, 5mM acetic acid) (Buffer 2- 40mM NaOAc, 40mM acetic acid) (Buffer 3-100mM NaOAc, 100mM acetic acid) Mobile Stationary • Lowering pH neutralizes negative charge on the protein molecules • Increase anionic molecules for competition 1 2 3 4 5 6 7

33. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Buffer II Buffer III Glucose oxidase Catalase

34. BY1101 Introduction to Molecular and Cellular Biology Ion exchange chromatography Add molecules of varying ionic charge Negatively charged molecules bind to beads Addition of different buffers changes charge

35. BY1101 Introduction to Molecular and Cellular Biology Functional assays In biological studies, what is the role of control samples? Catalase Glucose oxidase Control Glucose Hydrogen peroxide (H2O2) Control Glucose Hydrogen peroxide (H2O2)

36. BY1101 Introduction to Molecular and Cellular Biology Summary Chromatography: Used to separate out mixtures Affinity chromatography Gel filtration chromatography Ion exchange chromatography Separate molecules based on size Separates molecules based on charge Separates molecules based on biological specificity

37. BY1101 Introduction to Molecular and Cellular Biology MCQ Advice • Get your lab books up to date (e.g. Tables, graphs) • If you have problems with the questions ask a demonstrator (or me) • When it comes to the exam, revise all of the lab book (including the introductory notes) • Read over lab slides available on the teaching website

38. BY1101 Introduction to Molecular and Cellular Biology Next week - Developmental biology: Embryology • Lectures 3, 4 and 5 • Campbell: Chapter 47 (section 47.1)