I have recently been having trouble with protein digests from gels
clogging the HPLC columns I use for peptide mapping. Up to this point I
have mostly performed and separated digests off PVDF, but we recently
started doing digests in gel blots. My back pressures have sky-rocketed
and I just lost a $400 column because of it. I filter all my samples
before I inject then onto the HPLC (nylon, .45 micron) so I don't know what
is happening. Injecting THF across the column helped some, but still did
not bring the back-pressure down to an acceptable level. I suppose it is
possible that something in the sample is precipitating when it hits the
column (pH 8.5 to pH 2) but I haven't seen this before.
I am wondering how other labs prepare solutions from gel-digests
before they inject them onto the column. Is there something I am missing?
I welcome any literature references.
Thanks,
Bill
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Bill Enslow
Protein Sequencing Technician
Cornell BioResource Center
Rm 143 Biotechnology Building
phone: 607-254-4848
fax: 607-254-4847
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