Purified IgG on SDS-PAGE

Mike Clark mrc7 at cam.ac.uk
Mon Oct 2 05:29:54 EST 2000


In article <8r287l$4os$1 at oyez.ccc.nottingham.ac.uk>, Student
<URL:mailto:mucineer at iname.com> wrote:
> I 've recently run some antibodies (monoclonal) purified from tissue
> culture supernatant on a reducing SDS-PAGE gel (Novex) and stained by
> colloidal blue. On the heavy chain band, I notice that instead of one
> band, I have two very close bands, with the larger one fainter than the
> smaller one. The light chain, on the other hand, is one fine band.
> 
> Has anyone come across this? The source of my monoclonal was purified by
> protein A chromatography. Does the concentration I load onto the gels give
> this effect? What other possibilities are there?
> 
> Thanks for any input.
> 

The most likely explanation is heterogeneity in glycosylation of the heavy
chain. There is a conserved N-linked glycosylation site at Asn 297 in the
CH2 domain of IgG. In addition there is the possibility of V-region
glycosylation sites.

Mike's antibody structure and function website

<URL:http://www.path.cam.ac.uk/~mrc7/>

Mike
-- 
M.R. Clark, PhD. Division of Immunology
Cambridge University, Dept. Pathology
Tennis Court Rd., Cambridge CB2 1QP
Tel.+44 1223 333705  Fax.+44 1223 333875






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