1. Dr. A. Khaleel Ahamed Associate Professor of Botany
Jamal Mohamed College
Tiruchirappalli –
Mobile No

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 lipids:
>2% detergent
Precipitation
Removal of polysaccharides:
Removal of solid
material
Centrifugation
Removal of salts
Microdialysis
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. Protease inhibitors 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
Dialysis
Gel filtration
Precipitation/
resuspension
Slow
Protein losses
Complicated, can cause losses

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
IPG Buffer (carrier ampholyte mixture)
- 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 pH
7 cm, 13 cm and 18 cm strips:
pH L (linear gradient)
pH NL (non-linear gradient)

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

29. Guidelines for choosing Immobiline DryStrip 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, °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
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
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

51. Staining of Polyacrylamide Gels

52. References More help: ?? Amersham Pharmacia Biotech Handbook:
On the Internet:
Angelika Görg’s manual on her Website:
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