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Debora Fontanini 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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