alkylation with NEM and Hg treatment of protein

Achim Recktenwald, PhD achim at ibex.ca
Thu Feb 27 12:42:58 EST 1997


Michael O. Skidmore wrote:
> 
> Has anyone treated their protein with NEM to alkylate the sulfhydryls
> and reduce their microheterogeniety? It's been suggested I try this
> since there are 14 cysteines in our protein none of which appears to be
> in disulfide bonds.  I haven't been able to find much in the literature
> about this and would appreciate hearing about others experiences and any
> relevant reading. The lab that suggested it had excellent sucess with
> the one project they did it with. In our case, using DTT and/ or beta
> mercaptoethanol still results in some denaturation of the uncrytalized
> protein and a bad skin formation at 4 degrees .
> 
> I am also interested in hearing about pretreating your protein with
> stoichiometric ratios of HgCl2 and EDTA to prebind Hg before
> crystallisation.
> --
> Michael Skidmore                                  (619) 534-1894
> UC San Diego, Molecular Pathology
> 9500 Gilman Drive, Dept 0612
> La Jolla CA 90293-0612        http://www-acs.ucsd.edu/~mskidmor/


Check some older books - from the pre-genetic engineering era - about
chemical modification. I am not sure it's in there, but for example,
check the folowing reference I took from my PhD-thesis:
A.N. Glazer, et al. 'Chemical Modification of Proteins' in 'Laboratory
Techniques in Biochemistry and Molecular Biology', North-holland
Publishing, Amsterdam, 1976, vol. 4, Part 1.

Good luck,

Achim




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