IgG Coomassie staining - related problem

Student mucineer at iname.com
Tue Oct 31 08:09:57 EST 2000


I have recently purified two human monoclonal IgG (engineered) from
supernatant experiments by Protein A-Sepharose. When I ran them on SDS-PAGE,
I noticed two observations that puzzle me:

1) On the reduced gel, while the light and heavy chain bands are
approximately 25 and 50kDa, the light chain of one antibody is about 1kDa
smaller than the other antibody. Whilst I understand differences in protein
sequence may account for this, can the difference be so high (1kda)? Could
it be possible that one kappa light chain is glycosylated and the other is
not?

2) I then ran a non-reduced gel on the same purified antibodies. I found
that there are three main bands at around 110kDa to 150kDa, plus a range of
fainter bands below (various sizes). My question is this: even if there is
heterogenous glycosylation, can the size vary so much (from 110kDa to
150kDa)? and what are those other bands (at sizes down to 20kDa)? Can they
be fragments of the whole IgG that formed on heat denaturation? But if they
are, why aren't these seen in the non reduced gel (only two bands observed)
as well (heat denaturation period is the same)?

THanks for any insight.









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