Re: [ccp4bb] Crosslinking reagents

- Protein crystallography

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

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From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2008-03-04

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Subject: Re: Crosslinking reagents
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2008-03-04

Nian,

It is also important to point out that gluteraldehyde is quite
penetrating (which is why it is used as a fixative in EM) and
volitile. A 1% solution is 100 mM, which is quite concentrated.
Adding gluteraldehyde by vapor diffusion is quite effective and
gentle, but does take a bit of time (hours). Remember that
gluteraldehyde "fixes" by creating Schiff's bases. Thus, a non-
colored crystal turns a light gold color, which is the best optical
indicator of fixation. However, things will go awry if there are
lots of solutes around with free amines. The best example is Tris
buffer. So soak/grow your in mother liquor with no free amines other
that those on the protein. You can quench it with ammonium chloride
if desired.

Good luck,

Michael

****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: garavito@msu.edu
****************************************************************

On Mar 4, 2008, at 11:13 AM, David M Shechner wrote:

> Quoting Nian Huang :
>
>> I will try that. But I don't think people can tell the final
>> concentration of glutaraldehyde in the drop by using its vapor. Maybe
>> using direct soaking is what I should do. By the way, should I quench
>> it by ammonium or not?
>>
>> Nian Huang
>> Dept of Biochemistry
>> UT Southwestern Medical Center
>> Dallas, TX 75390
>
> Often it's the case that explicit knowledge of the gluteraldehyde
> concentration
> in the vapour isn't as important as empirical determination of the
> exposure
> time you subject the drop to, prior to moving it to a new well of
> non-crosslinking mother liquor. Here's the first (to my knowledge)
> reference
> that uses this technique; they mention using a time-course to
> determine when to
> quench:
>
> Lusty, CJ (1999) A gentle vapor-diffusion technique for cross-
> linking of protein
> crystals for cryocrystallography. J. Appl. Cryst. 32. 106-112.
>
> Cheers,
> d.s.
>
> ===========================================
> David M. Shechner, Graduate Student
> Bartel Laboratory
> Department of Biology
> Whitehead Institute for Biomedical Research
> Massachusetts Institute of Technology
> shechner@mit.edu
> ===========================================
>

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

Previous message:
Subject: Re: phenix.refine and refmac
From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2008-03-04

Next message:
Subject: Postdoc positions at Purdue University
From: "Chen, Jue" chenjue {- at -} PURDUE {- dot -} EDU
Date: 2008-03-04