2D-Electrophoresis is an important technique that is being used extensively in the Biochemistry and molecular biology for the quantification of different bio-molecules. It is also used in the different researches like cancer study etc. This presentation covers the introduction, sample preparation, main methodology and steps, staining techniques, applications, cost and availability across Pakistan. It also explains that why there is a need to replace the Electrophoresis with 2D electrophoresis. The main purpose of this effort is to highlight the main points about 2D-Electrophoresis.

1. 2D-
Electrophoresis
By: Husnain Tariq (Gold Medalist)
PhD Scholar (University of Gujrat)

2. Contents:
– Electrophoresis
– 2D-Electrophoresis
– Introduction
– Procedure
– Flow Sheet Diagram
– Applications

3. Electrophoresis
– is the migration of charged molecules in a liquid medium under
the influence of an electric field
– Three different types of supports are used:
1. Paper – amino acids, small peptides
2. Polyacrylamide – Proteins, small DNA/RNA
3. Agarose – DNA/RNA
(Dukhin & Goetz, 2002)

4. 2D-Electrophoresis
– separation and identification of proteins in a sample by
displacement in 2 dimensions oriented at right angles to one
another
– Isoelectric Point
– SDS-PAGE
Jefferies, et al

5. Why 2D-
Electrophoresis...???
To separate two proteins that have
same molecular mass

6. Introduction
– Firstly introduced by O’Farrell and Klose in 1975.
– Developed to separate the protein that have same molecular
weight.
– It separates thousands of protein simultaneously.

7. Sample preparation:
– Reagents used during sample preparation
1. 8M Urea
2. 100mM DTT
3. 4% CHAPS
4. 0.2% Carrier ampholytes
5. 40mM Tris
6. 0.002% Bromophenol Blue Dye
7. Water

8. Sample Preparation
– SAMPLE
– 40 mM Tris Supernatant 1
– Insoluble pellet 1
– 8M urea, 4% CHAPS, 100mM DTT
– 0.2% ampholytes, 40 mM Tris Supernatant 2
– Insoluble pellet 2
5M urea, 2% CHAPS,
– 100mM DTT
– 0.2% ampholytes, 40 mM Tris
– Supernatant 3

9. Isoelectric focusing
– Protein are focused to isoelectric point (pI), pH at which the net
charge on the protein is zero
– The net charge on a protein is the sum of all the negative and
positive charges of its amino acids
– Proteins have positive charge below their pI
– Proteins have negative charge above their pI
www.bio-rad.com

10. Isoelectric focusing
– +ve protein migrate towards cathode
– -ve protein migrate towards anode
– Separation is achieved by applying a voltage across a gel that
contain a pH gradient.
– Isoelectric focusing requires solid support such as polyacrylamide
gel
www.bio-rad.com

12. pH gradient:
– pH gradient is achieved by two ways:
1. Ampholytes
2. Immobilized pH gradient (IPG)
www.sciencedirect.com

13. Ampholytes:
– Firstly developed by Svensson in 1961
– Synthetic or natural
– Solubilizes the protein

14. Immobilized pH gradient (IPG)
– Firstly developed by Righetti in 1990
– generated by buffering acrylamide derivatives
(Immobilines)
– Immobilines are weak acid or weak base.
– The film-supported gel strips are easy to handle

16. SDS-PAGE
– IEF gel placed horizontally over the PAGE
– Separation is due to molecular mass
– SDS as surfactant
– Voltage is applied across the gel
– After staining, get the results
Lodish et al. Molecular Cell Biology

17. SDS
1. Denature protein
2. Negative charge to
protein

19. Staining:
– Different stains are used:
1. Coomassie blue – detect 36-47ng
2. Silver – detect 0.5-1.2ng
3. Fluorescent – detect 1-2 ng
Jefferies, et al

20. Softwares:
– BioNumerics 2D
– ImageMaster
– PDQuest
– Melanie
Arora et al., 2005

24. Cost & Availability
– Cost ranges from $1,121 to $1,524
– $1 = 104.82 pkr
– In Biochemistry and Molecular Biology Laboratries
– In NIBGE, CEMB, PU, QAU, UOL

25. Applications:
– Separation, identification, and quantitation of proteins
– Detection of post-translational modifications
– drug discovery
– cancer research
– purity checks
– micro-scale protein purification
Fey, et al., 2001

26. References:
– A.S. Dukhin & P.J. Goetz (2002). Ultrasound for the characterizing colloids, Elsevier.
– P.S. Arora, H. Yamagiwa, A. Srivastava, M.E. Bolander & G. Sarkar (2005).
Comparative evaluation of two-dimensional gel electrophoresis image analysis
software applications using synovial fluids from patients with joint disease, J.
Orthop. Sci. 10(2).
– Amersham Pharmacia Biotech - http://www.apbiotech.com.tw/
– http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.ht
ml
– http://www.sciencedirect.com/science
– http://www.bio-rad.com/en-pk/application