Jacob,
One of the problems with glutaraldehyde is the its chemistry is so
bizarre. It actually forms quite long transient polymers in
solution. You also have to ask yourself why formaldehyde also "fixes"
tissues. This is why glutaraldehyde works so well for tissue fixation
for EM as opposed to our usual bivalent crosslinkers we use in
biochemistry experiments. Check out the old EM literature about
discussions of glutaraldehyde chemistry.
Moreover, the Schiff's base linkage glutaraldehyde is slowly
reversible. You need to reduce it to make it permanent. I think that
glutaraldehyde is a very poor choice for a precise bivalent
crosslinker, but as a broad spectrum crosslinker (hitting lysines and
a free amino terminii that are different distances apart),
glutaraldehyde is great. As it is highly volatile (its unique smell
tells you you've had the bottle open too long), you can crosslink
crystals by vapor diffusion in an hour.
So I would be cautious about interpreting any crosslinking results
using glutaraldehyde, except the obvious (i.e., oligomers may indicate
the native tertiary state of a protein or complex).
Cheers,
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: rmgarav...@gmail.com
****************************************************************
On Sep 15, 2011, at 4:10 PM, Jacob Keller wrote:
Dear Crystallographers and Biochemists,
cross-linking, say with gluteraldehyde, is an oft-used method of
demonstrating a protein's oligomeric state in solution. I have a
difficulty with this, however: theoretically (and in practice!), one
can tune the amount of cross-linker to get what ever result is
desired, such that any protein with some exposed lysines can be
cross-linked in any oligomeric state. How, then, does one evaluate the
power of this evidence? Maybe one should do a gradient of
gluteraldehyde concentrations, then plot the deviation of the observed
cross-linked oligomerization from a theoretical null hypothesis? Seems
like this could be done, but I have never seen this in the
literature...
Best,
Jacob Keller
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
*******************************************
