Need help with IEF

David J. Meyer ez005587 at chip.ucdavis.edu
Thu Apr 22 10:48:50 EST 1993


gab at gnv.ifas.ufl.edu writes:
: 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

What *will* work very well with the mini tube gels is to run your
charge-based separation using NEPHGE (non-equilibrium pH gradient/gel
electrophoresis) rather than attempting IEF. This technique, which was
designed to separate basic proteins, worked rather well for the separation
of extensin, a pI >10 plant cell wall protein. In a Bio-Rad mini tube
apparatus, I found that electrophoresis at 500-750 V for 1 hour gave a
good separation. The original reference is:

O'Farrell, P.Z., H.M. Goodman, and P.H. O'Farrell. 1977. High resolution
two-dimensional electrophoresis of basic as well as acidic proteins. Cell
12:1133-1142.

I hope that this works for you; it's definitely worth a few experiments.

--
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David J. Meyer
djmeyer at ucdavis.edu



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