Dr. A. Khaleel Ahamed Associate Professor of Botany Jamal Mohamed College

1. Dr. A. Khaleel Ahamed Associate Professor of Botany Jamal Mohamed College Tiruchirappalli – 620 020 khaleeljmc@gmail.com Mobile No. +919443065561

2. Gel Electrophoresis An essential method to separate a mixture of charged molecules (DNA, proteins) Principle: migration in electric field and retardation by surrounding matrix Common matrices for protein gel electrophoresis: Agarose Polyacrylamide

3. 1D-Electrophoresis (SDS-PAGE) Denaturation and reduction Single amino acid chains Separation by molecular mass All proteins migrate towards anode

6. Applications of 2-D electrophoresis Analysis of cell differentiation Detection of disease markers Cancer research Purity checks and Microscale protein purification.

7. Experimental sequences for 2D electrophoresis 1. Sample preparation 2. IPG strip rehydration 3. IEF 4. IPG strip equilibration 5. SDS-PAGE 6. Visualization and 7. Analysis

8. Advantages of 2-D Electrophoresis Tolerant to crude sample loads: no pre-purification (like chromatography) has to be employed. ?? Highly resolution. ?? Very effective fraction collectors ?? Proteins are protected inside the gel matrix

9. Sample Preparation Cell washing ?? Cell disruption ?? Protein precipitation ?? Solubilization ?? Protection against protease activities ?? Removal of - nucleic acids - lipids - salts, buffers, ionic small molecules - insoluble material

10. Cell washing To remove contaminant material. Frequent used buffer - PBS (phosphate buffer saline): sodium chloride, 145 mM (0.85%) in phosphate buffer, 150 mM, pH7.2 - Tris buffer sucrose (10mM Tris, 250 mM sucrose, pH 7,2) Enough osmoticum to avoid cell lysis

11. Cell disruption methods Gentle lysis method 1. Osmotic lysis (cultured cells) ?? Suspend cells in hypoosmotic solution. 2. Repeated freezing and thawing (bacteria) Freeze using liquid nitrogen 3. Detergent lysis (yeast and fungi) Lysis buffer (containing urea and detergent) SDS (have to be removed before IEF) 4. Enzymatic lysis (plant, bacteria, fungi) Lysomzyme (bacteria) Cellulose and pectinase (plant) Lyticase (yeast)

12. Cell disruption (continued) Vigorous lysis method 1. Sonication probe (cell suspension) Avoid overheat, cool on ice between burst. 2. French pressure (microorganism with cell wall) Cells are lysed by shear force. 3. Mortar and pestle (solid tissue, microorganism) Grind solid tissue to fine powder with liquid nitrogen. 4. Sample grinding kit (for small amount of sample) For precious sample. 5. Glass bead (cell suspension, microorganism) Using abrasive vortexed bead to break cell walls.

13. Cell disruption (continued) Key variables for successful extraction from crude material 1. The method of cell lysis 2. The control of pH 3. The control of temperature 4. Avoidance of proteolytic degradation

14. Removal of contaminants Major type of contaminants: 1. DNA/RNA 2. Lipids 3. Polysaccharides 4. Solid material 5. Salt

15. DNA/RNA contaminant DNA/RNA can be stained by silver staining. They cause horizontal streaking at the acidic part of the gel. They precipitate with the proteins when sample applying at basic end of IEF gel How to remove: 1. Precipitation of proteins 2. DNase/RNase treatment 3. Sonication (mechanical breakage) 4. DNA/RNA extraction method (phenol/chroloform)

16. Removal of other contaminants • Removal of solid • material • Centrifugation • Removal of salts • Microdialysis • Precipitation Removal of lipids: >2% detergent Precipitation Removal of polysaccharides: Precipitation

17. Protein precipitation Ammonium sulfate precipitation: De-salting necessary TCA precipitation: Can be hard to resolubilize Acetone and/or ethanol: many proteins not precipitated TCA plus acetone: More effective than either alone, good for basic proteins

18. Protein solubilization Urea (8-9.8 M), or 7 M urea / 2 M thiourea Detergent (CHAPS,…) Reductant (DTT, 2-mercaptoethanol) Carrier ampholytes (0.8 % IPG buffer) Sonication can help solubilization

19. Reductants DTT (dithiothreitol) most commonly used DTE (dithioerythreitol) interchangeable with DTT 2-mercaptoethanol required at high concentration, contains impurities Tributylphosphine Poorly soluble, very hazardous Triscarboxyethylphosphine Good reductant, but negative charge makes it unsuitable for 1st dimension. Triscyanoethylphosphine Uncharged, soluble, but efficacy as reductant is in doubt.

20. Proteaseinhibitors PMSF (phenylmethyl sulfonyl fluoride) Most commonly used Inactivates serine and cysteine proteases AEBSF (Pefabloc) More soluble, less toxic than PMSF, but can cause charge modifications(?). EDTA Inhibits metalloproteases High pH Inhibits most proteases, but avoid Tris base

21. De-salting techniques • Slow • Protein losses • Complicated, can cause losses Dialysis Gel filtration Precipitation/ resuspension

22. FIRST DIMENSION

23. First Dimension: Denaturing IEF High molar (8 mol/L) urea, thiourea - one conformation of a protein - for protein solubility - prevents protein aggregates and hydrophobic interactions Non-ionic or zwitterionic detergent - for protein solubility IPG Buffer (carrier ampholyte mixture) - for protein solubility - raises the conductivity of the DryStrips DTT, DTE (no 2-mercaptoethanol) - prevents different oxidation steps

24. IEF with Carrier Ampholytes

25. Plot of the net charge of a protein versus the pH of its environment

27. Immobiline DryStrips: 1st Generation 11 cm strips: pH 4 - 7 pH 3 - 10 pH 6 - 11 7 cm, 13 cm and 18 cm strips: pH 4 - 7 pH 3 - 10 L (linear gradient) pH 3 - 10 NL (non-linear gradient) pH 6 - 11

28. Wide and Narrow pH Gradients Widegradients are applied for entire protein spectrum Narrowgradients are applied for: - increased resolution - increased loading capacity

29. Guidelines for choosing ImmobilineDryStrip gels

30. Ettan IPGphor

31. The IPGphor Platform

32. IPGphor Strip Holder

33. IPGphor features Platform accommodates up to 12 strip holders 7, 11 , 13 , 18 and 24 cm strip holders Cup-loading stripholders for all lengths Built-in power supply delivering 8000 V, 1.5 mA Built-in Peltier cooling, 18 - 25 °C Programmable “delayed start” rehydration period 10 possible programs, 10 phases each (ramp or step) Safety lid

34. Multiphor II

35. Buffer Tank

36. Cooling Plate

37. Safety lid

38. IPG Strip Reswelling Tray

40. Positioning IPG strips on Multiphor

41. Positioning the electrodes

42. Cup-loading of Sample

43. Two - Dimensional Electrophoresis

44. Principle of 2-D Electrophoresis 1. First dimension: denaturing isoelectric focusing separation according to the pI 2. Second dimension: SDS electrophoresis separation according to the MW The 2-D electrophoresis gel resolves thousands of protein spots.

45. Common reagents of PAGE Monomer: Acrylamide Basic unit in PAGE gel Neurotoxic Bridge: Bis, [N,N'-methylene-bis(acrylamide)] Cross-linker Neurotoxic Free radical generator: Ammonium persulfate Generation of free radical Riboflavin (vitamin B2) can also be used Catalyst: TEMED (Tetramethylethylenediamine) Assist transfer of electron of free radical

46. Choice of electrophoretic system

47. Choice of electrophoretic system

48. Second Dimension on Vertical Equipment

49. Staining Methods Colloidal Coomassie stain Fluorescent stain Coomassie stain Silver stain

50. Sypro Ruby protein staining 1. Simple protocol. No overstainng. 2. Less protein to protein variation 4. Stains glycoproteins, lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteins 5. Do not stain DNA/RNA 6. MS compatible 7. Expensive