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Monica Mittal

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Sep 4, 2009, 10:59:26 AM9/4/09
to prot...@magpie.bio.indiana.edu
HI
I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein but its showing a kind of hydrophobic behaviour as its max part is going into pellet after lysis.
moreover its not responding to ni2+ nta column also and the lysis sol. is not so clear that i go for gel filteration as it may probably clog it
so can u suggest any solution?

monica

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Dr Engelbert Buxbaum

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Sep 8, 2009, 9:58:37 AM9/8/09
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Am 04.09.2009, 10:59 Uhr, schrieb Monica Mittal <monicam...@yahoo.com>:

> HI
> I'M FACING PROBLEM IN PURIFYING A protein which is a dna binding protein
> but its showing a kind of hydrophobic behaviour as its max part is going
> into pellet after lysis.
> moreover its not responding to ni2+ nta column also and the lysis sol.
> is not so clear that i go for gel filteration as it may probably clog it
> so can u suggest any solution?

Does that mean you are expressing a poly-His tagged protein in bacteria?
If so, have you checked that your protein is not in inclusion bodies? How
did you lyse? How long and at what g did you try to clarify your lysate (1
h at 100000 g should do the trick)? Have you fragmented the bacterial DNA,
and are you sure that your protein is not binding to the fragments? Are
you protecting against oxidation and proteolysis?

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