Need help with IEF

gab at gnv.ifas.ufl.edu gab at gnv.ifas.ufl.edu
Tue Apr 20 14:47:53 EST 1993


Hello all!

I have just begun to do some isoelectric focusing and have run into
a snag.  I am using BioRad's mini-protean cell, which uses capillary
tubes for the IEF gels.  The directions that came with the kit called
for using 1.6% of ampholytes in the 5-7 pH range, and 0.4% of a broad-
range ampholyte, pH 3-10.  Since the protein I am working with has a
pI of about 10, I decided to only use the broad range ampholytes, and
bring their concentration up to 2%.  

The problem I have encountered is that when I checked the pH of the
gel (by cutting it into 10 pieces, putting each in a small volume
of .05 M KCL under vacuum for 2 hours, then checking the pH), I 
still only get a range of 4.3 - 8.

I would appreciate any suggestions on how to get a gel with a pH
gradient of 3-10.  Thank you in advance-    Beverly



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