Re: [ccp4bb] resuspending precipitated protein

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: SOme wquestions about refining.......
From: Kevin Cowtan cowtan {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2008-10-02

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From: "Mark J {- dot -} van Raaij" mark {- dot -} vanraaij {- at -} USC {- dot -} ES
Date: 2008-10-02

Subject: Re: resuspending precipitated protein
From: "Uitdehaag, J {- dot -} C {- dot -} M {- dot -} (Joost)" joost {- dot -} uitdehaag {- at -} SPCORP {- dot -} COM
Date: 2008-10-02

hi Andy

Here's my tip: If the protein is not denatured, but just precipitated, you can use the precipitate as research tool, trying to see if the protein will dissolve again with additives such as EDTA or divalent metal ions, or a bit of salt, or cofactor ATP or NAD, detergent etc (anything non-denaturing)

This will learn you about what the protein likes, and helps you to make a better prep that doesn't precipitate. We saw some miracles. If its rare protein and if it dissolves again you can even try to go ahead as if nothing happened,

best
Joost

Dr. Joost Uitdehaag
Dept. Molecular Pharmacology
group leader protein crystallography
Schering-Plough Research Institute, Oss
tel: +31-412-666738 fax: +31-412-662519

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Juergen Bosch
Sent: Thursday, 02 October, 2008 4:11
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] resuspending precipitated protein

6M Guanidiniumhydrochloride works pretty good, the question is only
will you be able to refold it correctly ?

I believe a better approach is to avoid precipitation in the first
place and optimize your purification procedure in such a way that it
works. When do you observe precipitate ? Dialysis, then us e a fast
desalting column instead.

Jürgen

On 1 Oct 2008, at 06:12, ANDY DODDS wrote:

> Hello,
>
> following on from a previous topic about precipitating protein, but I
> believe a distinct caveat of this warranting a separate thread, I
> would like to know people's experiences in trying to get precipitated
> protein back into solution? Is there a way or are there many ways?
>
> Any experiences would be welcome,
>
> yours,
>
> Andy

-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: SOme wquestions about refining.......
From: Kevin Cowtan cowtan {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2008-10-02

Next message:
Subject: Re: test data sets part II...
From: "Mark J {- dot -} van Raaij" mark {- dot -} vanraaij {- at -} USC {- dot -} ES
Date: 2008-10-02