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: [EMAIL PROTECTED]
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On Mar 4, 2008, at 11:13 AM, David M Shechner wrote:
Quoting Nian Huang <[EMAIL PROTECTED]>:
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
[EMAIL PROTECTED]
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