dfontani at facstaff.wisc.edu
Fri Jan 28 10:35:51 EST 2000
Hi Johan, hi everybody
Sorry for the partial explanation of my problem with the MonoQ; I'll try
to be more precise now.
The column volume is about 15 ml and I do not think that the gradient is
steep at all for this volume. I am going to 40% B in 30 minutes and my
sample comes off in the middle of the gradient.
When I was setting up the method for the column, I washed for 15 minutes at
40% and then at 100% for the same time: something comes off the column at
100% but nothing at 40%. Therefore, when I run the column for my sample
separation, I keep it at 40% for a couple of minutes and wash with 100%
every once in a while (the column has a very high capacity and I am
injecting very little protein amounts - around hundred of µg) just to get
off possible bound material.
The elution is monitored with a diodearray detector. The peak I collect is
the enzyme I am puryfing so I measure it with a proteolytic assay after the
collection. I doubt I introduced in the column something that might
degrade/digest my enzyme, since the only other samples I injected, are
enzymes I co-purified with it.
I hope I was detailed enough for anybody to come up with any help; I have
to admit I have no idea of what the problem could be.
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