[Protein-analysis] Re: QUERY

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Tue Sep 8 08:58:37 EST 2009


Am 04.09.2009, 10:59 Uhr, schrieb Monica Mittal <monicamittal41 from 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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