From: Jacob Keller
Date: 15 September 2011 21:10
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
----------
From: Ed Pozharski
Date: 15 September 2011 21:18
Right - just do it side-by-side with a protein known to be monomeric of
roughly the same size/lysine content... And what is the "critical
concentration" of gutaraldehyde at which the false positives appear in
your experience?
--
"I'd jump in myself, if I weren't so good at whistling."
Julian, King of Lemurs
----------
From: Jacob Keller
The critical concentration depends on protein concentration, time of
reaction, brand of gluteraldehyde, day of week, color of my shirt....
No, I don't know--I have seen cross-linking gradients in Nature and
such in which several oligomeric states can be seen up to the one the
author asserts is the physiological one. This is a nice experiment for
proving one's point on paper, but maybe not for establishing the
truth? Maybe a control with some SDS would be appropriate (although
this would probably perturb the lysines). Or maybe the experiment
should be done in a lysate, and then western-blotted?
Jacob
----------
From: Herwig Schuler
Dear Jacob,
agree, it's a mess. From what I read, the glutataldehyde concentration should be low (<0.01%) and the x-linked complex that you get should not occur in high salt conditions (reasoning that 1.2M KCl would break the average complex apart). Have seen papers where more selective zero length crosslinkers have been used - the Pierce catalog used to have lots of them - it seems it boils down to the same problem, eventually you will find one that "works" but you will need independent evidence to convince yourself.
I typically make really nice MW size ladders with my monomeric negative control proteins, though.
Best, Herwig
* * * * * * * * * * * * * *
Herwig Schüler, PhD
PI, Structural Biochemistry
Structural Genomics Consortium, MBB
Karolinska Institutet
Scheeles väg 2
S-17177 Stockholm
----------
From: R. M. Garavito
Date: 15 September 2011 21:10
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
----------
From: Ed Pozharski
Date: 15 September 2011 21:18
Right - just do it side-by-side with a protein known to be monomeric of
roughly the same size/lysine content... And what is the "critical
concentration" of gutaraldehyde at which the false positives appear in
your experience?
--
"I'd jump in myself, if I weren't so good at whistling."
Julian, King of Lemurs
----------
From: Jacob Keller
The critical concentration depends on protein concentration, time of
reaction, brand of gluteraldehyde, day of week, color of my shirt....
No, I don't know--I have seen cross-linking gradients in Nature and
such in which several oligomeric states can be seen up to the one the
author asserts is the physiological one. This is a nice experiment for
proving one's point on paper, but maybe not for establishing the
truth? Maybe a control with some SDS would be appropriate (although
this would probably perturb the lysines). Or maybe the experiment
should be done in a lysate, and then western-blotted?
Jacob
----------
From: Herwig Schuler
Dear Jacob,
agree, it's a mess. From what I read, the glutataldehyde concentration should be low (<0.01%) and the x-linked complex that you get should not occur in high salt conditions (reasoning that 1.2M KCl would break the average complex apart). Have seen papers where more selective zero length crosslinkers have been used - the Pierce catalog used to have lots of them - it seems it boils down to the same problem, eventually you will find one that "works" but you will need independent evidence to convince yourself.
I typically make really nice MW size ladders with my monomeric negative control proteins, though.
Best, Herwig
* * * * * * * * * * * * * *
Herwig Schüler, PhD
PI, Structural Biochemistry
Structural Genomics Consortium, MBB
Karolinska Institutet
Scheeles väg 2
S-17177 Stockholm
----------
From: R. M. Garavito
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
****************************************************************
No comments:
Post a Comment