PROBLEM RESOLVING H & L chain intermediates on gel

Oh Kah Weng Steve engohkw at leonis.nus.sg
Tue Dec 13 00:58:01 EST 1994


Dear surfers,

We have been trying for several months to resolve antibody 
intermediates harvested from hybridoma cell extracts to measure their 
quantities under different conditions. However, when we run the 
non-denaturing gel, we have problems getting good separation between the 
H2-L2, H2-L and H2 molecules. They are all crammed up near the top of the 
gel occupying a space less than 1cm.....like so:-

                      Bands     Molecules    M.W.

                      _____     H2-L2        150
                      -----     H2-L         125
                      -----     H2           100

QUESTION: Does anyone know of a good method to get better separation, say 
over a 2-3 cm distance between all these intermediates?

OUR CONDITIONS: 4% acrylamide stacking gel, 
                8% resolving gel, 
                Running buffer has 0.25M Tris and 1.92M Glycine with 10%SDS.

If anyone out there think they can help but need more information please 
respond directly to engohkw at leonis.nus.sg. Thanks a lot.

P.S. Not only are we trying to resolve these bands but we hope to 
quantitate the amounts too between samples (a high and a low 
antibody producer)!!! The scientists that we have talked to 
here says that this is almost impossible. (Hope someone out there knows
different)    THANKS AGAIN!!!



..................AND A MERRY CHRISTMAS TO ALL!!!



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