gelfiltration on superdex 75 pg
ffrank at rz.uni-potsdam.de
Fri Sep 29 06:41:39 EST 2000
Sigrid Van Boxstael wrote:
> When I clean the column after the run there is coming protein of it.
> I presume that my protein sticks on the column.
> I think that increasing the salt concentrations might be the solution.
Probably yes, as the others have written. But there's an other
possibility: Specific binding to the column if the protein has some sort
of sugar binding activity.
I'm working with a protein that specifically, but rather weakly (Kd=1 to
2 mM) binds glucose and mannose, and there's also a mutant (point
mutation) that lacks binding affinity, as judged by published data or by
every method so far used in our laboratory. But:
I tried to use a small column hand-made of Superdex 200 pg material as
an affinity column to quantify the amount of active protein.
Unfortunately, I noticed that binding to the column is much stronger
- With the wild type, I don't get elution with saccaride free buffer (as
expected), with 1 M glucose I get most of it off the column. But then I
give a short pulse of NaOH and get one more peak, and when I repeat the
procedure without injecting new protein, I can again elute one small
peak with glucose and one with NaOH.
- About half of the "inactive" mutant elutes as on a normal gel
filtration column, but a significant part only comes off with glucose,
and an other part with NaOH.
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