determining oligomeric structure of a protein

[Jesus M. Sanz]

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 
> machine.
> 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
useful
where dimethyl suberimidate and glutaraldehyde cannot work. You can find
a nice review about bifunctional reagents in Methods Enzymology (91), 580
(1983).

Cheers
-- 
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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