determining oligomeric structure of a protein
Jesus M. Sanz
sanz at mrc-lmb.cam.ac.uk
Sat Mar 5 07:02:07 EST 1994
In article <2l675s$mna at charm.magnus.acs.ohio-state.edu>,
rsaldanh at magnus.acs.ohio-state.edu (Roland J Saldanha) wrote:
> I am working on the mitochondrial tyrosyl synthetase that functions as both a
> synthetase and as a facilitator of group I intron splicing. In characterizing
> the protein we find that it elutes from gel filtration columns with an apparent
> molecular weight of a tetramer (monomer MW=72,000). However the protein
> crosslinks as a dimer with dimethyl suberimidate and glutaraldehyde. This
> would suggest a highly elongated structure that has an aberrant mobility
> (reflecting the elongated size) in gel filtration.
> I would welcome suggestions for other methods for demonstrating the oligomeric
> structure of the protein. I am aware that sedimentation analysis is the best
> method of approaching this problem but do not have access to an analytical
> ultracentrifuge and would welcome collaborative offers from anyone who has a
> We would also like to try native page but the protein is very basic (computer
> calculated pI of 10.3). I would thus welcome suggestions for native gels of
> basic proteins and suitable standards for basic proteins.
You could also try another crosslinking reagent with a different
specificity. For instance, water-soluble
carbodiimides are good for crosslinking between carboxylates, and may be
where dimethyl suberimidate and glutaraldehyde cannot work. You can find
a nice review about bifunctional reagents in Methods Enzymology (91), 580
Dr. Jesus M. Sanz Tf. (0223) 402146
Centre for Protein Engineering. Fax.(0223) 402140
Hills Road, e-mail:sanz at mrc-lmb.cam.ac.uk
Cambridge CB2 2QH, U.K.
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