Problem with Isoelectric Focusing on gels

Christian Collin-Hansen Christian.Collin-Hansen at chem.ntnu.no
Tue Jul 8 07:51:48 EST 2003


Wo, thanks for your quick responce!


If I understand you correctly, the TCA fixing step is done to
"preipitate" the proteins within the gel. This protein is soluble in
0% (pure water) to well above 30% TCA. There are, however, some loss
at the higher TCA concentrations, so I don't go above 10%.


I have tried  HIC with 4M NaCl to salt it out of solution and onto the
coloumn, but it passed strait through. Thus, I guess high salt conc.
can be used as an alternative to TCA as an initial step in the
purification process (leaving the protein in the supernatant just like
with TCA). The reason for using TCA is that it makes a lot of other
stuff presipitate that would probably not be presipitated by salt
alone. Anyhow, it still does not solve the problem of fixing the gel
in TCA...


So, I seem to be stuck with the same problem: I can't fix the gel in
TCA prior to staining and I can't leave the step out, since both would
cause the protein to come out of the gel. At least: Now I know!


Do you know of any other easy way of determining the isoelectric point
of proteins?



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