couple Inclusion bodies on sepharose?
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Feb 1 18:52:18 EST 1999
In article <36B02F66.2ED2BE9B at uni-muenster.de>, "Andreas Vogel" <vogela at uni-muenster.de> wrote:
:I would like to affinity purify rabbit sera against a protein which I
:isolate as overexpressed Inclusion bodies from E.coli. Is it possible to
:couple the IB╢s on activated CH-sepharose?
:Any hints are welcome,
Well not just add serum to inclusion bodies? Then you just spin down,
resuspend in elution buffer (i.e. low pH), then spin down again.
Et voila! (In an unlikely case that IBs partially dissolve at low pH,
this won't work too well, but the same is true for sepharose coupled
IBs; then 3.5 M MgCl2 buffered to pH 6-7 might work).
ALternatively, you can dissolve IBs in SDS and couple to just about
anything in the presence of SDS. The problem here is that upon removal
of SDS, you are likely to end up with randomly folded, highly hydrophobic
polypeptides linked covalently to the sepharose. Blocking first
will become almost obligatory.
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