Help with large SDS-PAGE gels

Simon Twigger mbxsnt at unicorn.nott.ac.uk
Mon Jul 4 07:32:14 EST 1994


Another method I have found to give excellent results without all the
hassle involved in the Schagger and G. von Jagow method is the use of a
High Tris buffer system.

I typically use 10-20% gradient gels (19cm x 11cm) and get excellent
resolution down to 8kDa and below. The method is taken from:

Fling, S.P. & Gregerson, D.S. Analytical Biochem 155:83-88 (1986)

They are easy to cast, take about 4-5hours to run at approx. 350V (Constant
voltage with cooling) and I get some really good results from them. We have
also used this system for mini-gels (no gradient) and find that there is an
improvement over the standard Laemelli system. One small problem is that it
does use quite a lot of Tris!

I have also tried a trick I saw mentioned in a paper which the authors
atributed to a Pharmacia leaflet and that was to use 25% Ethylene glycol in
the resolving gel. This gave good results for proteins 20kDa and below (on
20% gels) but proteins larger than that did run a bit odd.

Hope this might help.

    Simon.


==============================================================================
Simon Twigger                                  =  
University of Nottingham Biochemistry Dept.    =  
E-MAIL     mbxsnt at uk.ac.nott.unicorn           = 
           livvy at wings.micro.umn.edu           =
==============================================================================



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