2-D Electrophoresis

In 2-D electrophoresis, proteins  are separated in the first dimension, according to their isoelectric points, by IEF, and in the second dimension, according to their molecular weights, by SDS-PAGE24,25. The first, IEF, stage is conducted in a long, thin,  spaghetti-like gel.  This gel must be of large pore-size so that focusing is possible, but this makes it very soft and fragile. For the  second stage the  long, thin,  gel is transferred  to  the origin of a slab gel,  sealed in position  and an  SDS-PAGE separation  is carried out.  The  result,  after  staining,  is  a  number  of  spots  distributed  in two dimensions over the slab gel.  Because the first-stage gel is of a small diameter, only a small amount of ampholytes is consumed per run and so 2-D electrophoresis is relatively inexpensive.

 Although conceptually simple, it took  a number of years for the method to be developed to its present stage of practicability.  One  of the technical difficulties to be overcome was the handling and transferal of the first-stage gel  soft spaghetti is not the easiest type  of material to handle!

References 

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .

Garrels,  J.  I.  (1 983)  Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol.  100, 41 1-423.

Dunbar, R. S., Kimura, H. and Timmons, T. H. (1990)  Protein analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis. Methods Enzymol. 182, 441-459.

مواضيع ذات صلة

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production

Introduction to Immunological methods

Isoelectric focusing

Pore gradient gel electrophoresis

SDS-PAGE

Polyacrylamide gel electrophoresis

Starch gel electrophoresis

Agarose gel electrophoresis