dfontani at facstaff.wisc.edu
Thu Jan 27 21:46:02 EST 2000
I have a problem and hope some of you might have a suggestion or two on how
to solve it.
In the process of puryfing a peptidase, I have been separating my partially
purified protein sample on a MonoQ column. Buffer A is 20 mM TEA/HCl, pH
7.5 and B is A + 1M NaCl. The gradient goes from 0 to 40% B in 35 minutes.
For a few injections I was able to collect a peak coming off the column at
about 20% B. Then I changed the buffer system to separate a different
sample and when I switched back to the original system and sample, nothing
would come off the column. Thinking that the column was dirty, I cleaned
it following the manufacturer's instructions (not up to the point of using
pepsin, yet) and despite an improvement in the baseline, there was no
improvement in the sample recovery.
I might add, if it is of any help, that the column is fairly new (used for
about 10 injections) and I am only injecting 150 µl of sample at the time
since I am using an HPLC system adapted to work as FPLC and I do not have a
larger loop available.
I would greatly appreciate any suggestion
Thanks in advance,
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