Problem with Isoelectric Focusing on gels

Christian Collin-Hansen Christian.Collin-Hansen at chem.ntnu.no
Fri Jul 4 07:58:36 EST 2003


Problem: I am trying to find a method for isoelectric focusing (IEF)
of a protein that is soluble in 10% tricloroacetic acid (TCA). An
early step in the purification  process for this protein is to add 10%
TCA to the sample, and the protein remains within the supernatant.

It appears to be a conventional practise after IEF-run by gel to fix
the gel i 10-12% TCA prior to staining  (e.g. the IEF-gels for the
Phastgel-system of Amersham or the Novex IEF-gels from Invitrogen). By
IEF on Phastgel no band is visible after silver staining, in contrast
to nice bands from the standards. I fear that the protein may have
come out of the gel during the post-run TCA fixing step.

Question: Is it nessecary to fix IEF-gels in TCA after focusing (prior
to staining)? Are there alternative ways of fixing the gel?



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