Dennis,
You could try the following:
If the sequence of each of the proteins is known, determine the theoretical
tryptic peptides that would be generated for each of the proteins.
Presumably with an approximate 3000 Da difference you should expect to find
at least 1-2 peptides from the larger protein that are not present in the
small protein.
Using one of your antibodies (which recognizes both proteins)
immunoprecipitate the expressed proteins. I would recommend using SDS-PAGE
to verify the purity of your immunoprecipitate. If as you stated, the two
proteins can not be resolved by SDS-PAGE (although a difference of 3000 Da
should be adequate for resolution provided no complications are present due
to post-translational modifications) then you could cut out the SDS-PAGE
purified band or bands and complete an in-gel digestion with trypsin.
Using mass spectrometry (MALDI-TOF) look for the mass of the two C-terminal
peptides (peptide mass fingerprinting).
As an alternative approach (depends greatly on what the predicted
C-terminal peptides are for each protein) you could selectively isolate the
C-terminal peptides from you immunoprecipitated protein(s) using
anhydrotrypsin affinity chromatography. See Chait, Brian T. and Salvatore
Sechi Anal. Chem. 2000 Vol 72, 3374-3378 and see also Shin-ichi Ishii,
Hideyoshi Yokosawa, Takashi Kumazaki, and Izumi Nakamura Methods in
Enzymology, Vol 91, 378.
Hope this helps,
Cheers
Raymond Boynton
N-terminal Protein Sequencing Facility
Biogen, Inc.
Cambridge MA 02142
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