QUERY

[Monica Mittal]

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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[wajahat mahmood]

Hi Monica,

If your protein is in the pellets I hope you are using denaturing conditions to solubilize it before purification over Ni-NTA.

solubilize the protein in urea or guanidine for one hour and spin down to get a clear lysis solution before you proceed to further purification steps Ni-NTa or gel.

 

cheers


wajahat

> Date: Fri, 4 Sep 2009 20:29:26 +0530
> From: monicamittal41@...
> To: proteins@...
> CC:
> Subject: [Protein-analysis] QUERY
>
> 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
>
>
>
> Love Cricket? Check out live scores, photos, video highlights and more. Click here http://cricket.yahoo.com
> _______________________________________________
> Proteins mailing list
> Proteins@...
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[Dr. Yogendra Sharma]

its not going into pellet due to hydrophobicity. It is forming inclusion bodies during overexpression.
You are required to dissolve the protein and refold it.
Please fiollow the procedure "how to refold protein from inclusion bodies"
Good luck with your work
Yogendra
-- Original Message --
From: Monica Mittal <monicamittal41@...>
To: proteins@...
Date: Fri, 4 Sep 2009 20:29:26 +0530 (IST)
Subject: [Protein-analysis] QUERY
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]

Am 04.09.2009, 10:59 Uhr, schrieb Monica Mittal <monicamittal41@...>:

> 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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