studying dimerisation

Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands sbtnfh at rulsfb.LeidenUniv.nl
Mon Sep 12 10:08:24 EST 1994


Hello Netters,

I'm looking for a sensible and reliable method to find out if a protein is
a monomer or a dimer. For gelfiltration analysis you need a reasonable amount
of relatively pure protein, which I haven't got. I've mutagenized pea lectin
which is a dimer in order to try to get monomers. pea lectin is an all-beta
sheet protein, and the two subunits are "zipped" together by combining two
sheets. I express both wild type and mutant pea lectin in tobacco. The 
expression level is something like 1% of the total soluble protein in leaves
I've tried to use Dimethyl Suberimidate to cross-link the subunits, so I can
see the difference between monomers and dimers on a SDS-PAGE, (reference
Davies et al, PNAS 66: 651-656, 1970) but this doesn't work, not even with
wild type lectin. 
	Does somebody know of a method to cross-link subunits without having
to purify the protein first? Or maybe another method to study the subunit
structure?

Thanks, Flip

p.s. I meant sensitive and not sensible, but I have a lousy editor so I can 
not go back for typos Sorry



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