crosslinking

bford barry.ford at drdc-rddc.gc.ca
Thu Sep 1 12:27:54 EST 2005


In article <df6biu$6uj$1 at news.net.uni-c.dk>, nospam at nospam.com says...
> I want to study whether my protein on the surface of a cell is present as a 
> homodimer! I will do this by crosslinking the proteins and running a wb. 
> However there are so many crosslinking reagents, and I am not sure which to 
> choose. Does anyone have any suggestions.
> Thanks
> AS
> 
> 
> 
I used glutaraldehyde crosslinking a while back for assessing whether 
mutant porteins could from dimers.  It has the virtue/caveat of being 
relatively non-specific, but tends to link only proteins that are close 
together, as in homodimers, rather than in solution. The recipe is 
robust and highly adaptable. 

To an aliquot (5-10 mg) of protein in a total volume of 100 ml, 13 ml 1 
M borate pH 8.9 were added, followed by 8 ml 0.2 M glutaraldehyde (final 
glutaraldehyde concentration 14 mM).  At intervals of 0, 20, 60, 180, 
540 seconds, 10 ml of 1 M NaBH4 were added with rapid mixing to stop the 
reaction.  Crosslinking reaction productst were stored on ice until 
tracking dye was added and samples prepared for electrophoresis.  

Have fun. 

some refs;
Wu, B., A. Wawrzynow, M. Zylicz and C. Georgopoulos (1996) Structure-
function analysis of the Escherichia coli GrpE heat shock protein, EMBO 
J. 15, 4806-4816.

Cheung, D.T. and M.E. Nimni (1982) Mechanism of crosslinking of proteins 
by glutaraldehyde II. Reaction with monomeric and polymeric collagen, 
Connect. Tissue Res. 10, 201-216.


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