In addition to the info replied earlier:
The kinetics of photocrosslinking event itself, after a molecule is
photo-activated, is very fast (millisecond range). Since protein-protein
interactions are usually equilibrium conditions, proteins remain in
tight contact only a fraction of the time. And, even in case of very
high affinity complexes and slow off-rates, there are thermal
fluctuations even if proteins remained in contact and did not
dissociate. If a moiety being crosslinked happens to be photoactivated
at the moment when it is not in a very tight contact with it's potential
target, then crosslinking event is wasted to surrounding water without
useful analytical outcome.
Cooling down the sample from physiological temperature to ice
temperature slows down kinetics of protein interaction events (whatever
they are) by an order of magnitude, consequently increasing overall time
proteins remain in tight contact when crosslinking can occur. This
results in a noticeably increased yield of cross-link formation.
================================
Dmitry Bochkariov, Ph.D.
Principal Scientist
Advansta Inc.
1505 Adams Drive, Suite B1
Menlo Park, CA 94025
(650) 325-1980 x530
================================
On 12/6/2016 9:54 PM, DK wrote:
In article <mailman.111.1480644299.7738.meth...@net.bio.net>, Hiranya Roychowdhury
<hroyc...@nmsu.edu> wrote:
To minimize any degradation during the process, I would assume.
Also, a typical photo-crosslinking requires high intensity lamp and has
relatively slow
kinetics. When the sample is small, it can get overheated (and proteins
denatured)
in no time.
DK
Hiranya S. Roychowdhury, Ph.D.
Professor
Department of Science
Division of Science, Engineering and Math
NMSU-Dona Ana Community College
575 527 7725 (office)
Chair, Curriculum & Instruction Committee
Human Anatomy and Physiology Society<http://www.hapsweb.org/>
[http://www.hapsweb.org/graphics/header.jpg]
________________________________
From: methods-boun...@oat.bio.indiana.edu <methods-boun...@oat.bio.indiana.=
edu> on behalf of Sudheer Sangeetham <sudheer.pb...@gmail.com>
Sent: Thursday, December 1, 2016 10:18:38 AM
To: meth...@oat.bio.indiana.edu
Subject: UV 365 nm, Photo crosslinking on ice?
Hi everyone
I have checked many articles. All were done photocrosslinking on ice to
detect protein protein interactions or dimer formation? why not at room
temperature?
please let me know if anyone knew about it.
Thank you
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