Posted by jayaram in Principle
This technique combines the technique of IEF, which separates proteins in a mixture according to charge isoelectric point, with the size separation technique of SDS-PAGE. When combined to give 2D PAGE, the most sophisticated analytical method for separating proteins available is obtained.
Procedure
First dimension electrophoresis is carried out in polyacrylamide gels in narrow tube in presence of ampholytes, 8M urea and non-ionic detergent (ampholytes)
Therefore the denatured proteins separate in this gel according to their isoelectric point
The gel is then removed from the tube by applying slight pressure to one end.
The gel is then incubated for 15 min in a buffer containing SDS so that SDS binds to denatured proteins.
It is then placed on sacking gel and fixed in place by pouring molten agarose.
Once the agarose has set, electrophoresis is commenced and SDS bound proteins run into the gel stack and separate according to size.
Application
The characteristics of protein separated by 2D can be found by sequence analysis of protein. The sequence data is obtained by 2 ways.
Direct protein sequencing of spot using edman degradation
The proteins in 2D gel are electro blotted onto a suitable membrane like polyvinyl difluoride after staining of the blot; the spot of interest is excised and introduced directly into an automated sequence analyzer thus generating an amino acid sequence from N- terminus of protein.
Peptide mass profiling
Peptide mass obtained by mass spectrometric analysis of protein digest provides characteristic finger printing of protein.
The protein spot is digested within the gel matrix. The peptides are extracted. Theses unfractioned peptides are then analyzed using MS to give accurate molecular mass of peptide. This is then compaired with the database.