brief

1. ELECTROPHORESIS
BY
Pintu Choudhary
Master of Technology
( Food Science and Technology)
Indian Institute of Crop Processing Technology
Ministry of Food Processing Industries
Government of India
Thanjavur - 613005

2. Electrophoresis
• Electrophoresis is a method whereby charged molecules in
solution, chiefly proteins and nucleic acids, migrate in response
to an electrical field.
• As an analytical tool, electrophoresis is simple, rapid and highly
sensitive.
• It is used for analysis and purification of very large molecules
(proteins, nucleic acids) for analysis of simpler charged
molecules (sugars, amino acids, peptides, nucleotides, and
simpler ions).

3. Basic Principle
1. It is the process of moving charged
biomolecules in solution by
applying an electrical field across
the mixture.
2. Biomolecules moved with a speed
dependent on their charge, shape,
and size and separation occures on
the basis of molecular size.

4. Principle
When charged molecules are
placed in an electric field, they
migrate toward either the
positive (anode) or negative
(cathode) pole according to
their charge.

5. Factors Affecting Electrophoretic
Mobility
Number of parameter of electrophoretic system:
1. For zone EP, type of support medium chosen, and if it is a
gel, its pore size
2. Ph. of the electrophoresis buffer
3. Ionic composition of buffer
4. Applied voltage
5. Temperature

6. Supporting Medium
Selection is based on following consideration
• Size of the molecules to be analyzed
• Quantity of sample available
• Cost of support medium to be used
• Availability of suitable equipment
• Purpose of the analysis
• Time to run the analysis
• Expertise of the operator

7. Types of Electrophoresis
• Affinity electrophoresis
• Capillary electrophoresis
• Dielectrophoresis
• DNA electrophoresis
• Electro blotting
• Electro focusing
• Gel electrophoresis
• Immunoelectrophoresis
• Isotachophoresis
• Pulsed field gel electrophoresis

8. • Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
• High ionic strength of buffer
1. produce sharper band
2. produce more heat
• Commonly used buffer
1. Barbital buffer & Tris-EDTA for protein
2. Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
Buffers

9. Protein Electrophoresis
• Sample preparation
• Two-dimensional electrophoresis
• Detection of spots
• Image analysis
• Spot excising
• Enzymatic digestion of proteins
• Mass spectrometry
• Bioinformatics

10. 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

11. 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

12. Cell Disruption Methods
Gentle lysis method
1. Osmotic lysis (cultured cells)
– Suspend cells in hypo-osmotic 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)

13. Cell disruption (continued)
Key variable 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. Protein Precipitation
• Ammonium sulfate
(salting out)
• TCA precipitation
• Acetone and/or
ethanol
• TCA plus acetone
• Not efficient, de-
salting necessary
• Can be hard to
resolubilize
• Leaves SDS
behind, but many
proteins not
precipitated
• More effective
than either alone,
good for basic
proteins

16. 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
• Sample can be heated only prior to addition of
urea

17. Staining Methods
• Colloidal Coomassie stain (Gel Code Blue
from Pierce)
• Fluorescent stain (Molecular Probes)
– Sypro Ruby
– Difference Gel Electrophoresis (DIGE)
• Coomassie stain
• Silver stain

18. 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE)

19. Protein Identification by MALDI-TOF

20. SDS-PAGE
(Sodium dodecyl sulphate polyacrylamide gel electrophoresis)

21. Procedure
• Protein sample is first boiled for 5 mins. in a buffer
solution containing SDS and β-mercaptoethanol
• Protein gets denatured and opens up into rod-shaped
structure.
• Sample buffer contains bromophenol blue which is
used as a tracking dye, and sucrose or glycerol.
• Before the sample is loaded into the main separating
gel a stacking gel is poured on top of the separating gel.

22. Procedure continued…
• Current is switched on.
• The negatively charged protein-SDS complexes now continue to
move towards the anode.
• As they pass through the separating gel, the proteins separate,
owing to the molecular sieving properties of the gel.
• When the dye reaches the bottom of the gel, the current is turned
off.
• Gel is removed from between the glass plates and shaken in an
appropriate stain solution.
• Blue colored bands are observed under UV rays.

23. Choice of Electrophoretic System

24. Choice of Electrophoretic System

25. Zonal Electrophoresis

26. Application of Electrophoresis
• Clinical diagnosis
• SDS –PAGE : used for finding molecular weight of proteins and
their purification.
• Separation and analysis of nucleotides and nucleic acids, DNA
finger printing.
• Hemoglobin separation
• Lipoprotein separation and identification
• Determination of molecular weight of proteins.

27. References
• Dunbar, B. S. (1987). Two Dimensional Electrophoresis and
Immunological Techniques. New York: Plenum Press.
• Gersten, D. M. (1996). Gel Electrophoresis (Essential Techniques Series).
New York: Wiley.
• Kuhr, W. G. (1990). Capillary Electrophoresis. Anal Chem 62:403R–414R.
• Laemmli, U. K. (1970). Cleavage of Structural Proteins during the
Assembly of the Head of Bacteriophage T4. Nature 227:680.
• Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
• Tal, M., Silberstein, A., and Nusser, E. (1980). Why Does Coomassie
Brilliant Blue Interact Differently with Different Proteins? J Biol Chem
260:9976–9980.Press.