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vertical Isoelectric focusing - cathodic drift and plateau formation of pH gradient (Mar/02/2007 )


Im working on characterizing extended spectrum beta lactamases based on isoelectric focusing. I do vertical slab gel with the following composition - 7%T acrylamide plymer, 10%glycerol, 2% biorad ampholine 3-10.
Electrolytes - 20mM NaOH and 20mM orthophosphoric acid. My focusing conditions - 5000 volt-hrs at 500 volt constant. gel length is 13cm.
With these condition Im encountering problems like cathodic drift ( pH range above 7.0 is missing) and plateau formation between 5.0-6.5 pH range, and also poor protein separation.

how to avoid this kind of problem? should the electrolyte concentrations to be changed or focusing conditions to be altered??



your electrolyte concentrations may be a little low, i think i was using 0.1M (it may have been 1M, it was a long time ago). we also used a 5% acrylamide gel. however, you can never eliminate cathode drift entirely, you can only reduce it and hopefully end the run before the drift is too severe.

you can eliminate the plateau by the addition of a little 4-7 or 5-8 ampholyte.

of course, you can eliminate all of these problems if you use an immobilized pH gradient.