Heavy/light chains on Coomassie gel???

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Nov 1 10:06:03 EST 2000


Neal Robert Melvin <nrmelvin at ucalgary.ca> wrote:
:Hello,
:
:I've been using Coomassie Blue staining to get a rough idea of the
:concentration of normal rabbit serum. When I do this, I notice that the
:bands corresponding to the heavy chains stain significantly more intensely
:than the light chain bands... should these be the rough the same intesity
:since one IgG contains 2 heavy chains and two light chains??

Of course not. Heavy chains are twice larger than light and that 
means they will bind roughly twice more Coomassie.

:I'm trying to match the concentration of this normal rabbit serum to a
:known amount of immune rabbit serum so that I can perform an
:immunoprecipitation with equivalent amounts of IgG's... which bands should
:I try to match in intensity (between my immune serum and my non-immune
:serum), the heavy chains of each, or the light chains??

IMHO, none. If you want clean and reliable results, you should work
with purified IgG and measure their concentration directly. BTW, 
"non-immune" IgG control is really a very poor control. 
Even putting aside differences between rabbits (which 
could be dramatic as one can see from screening preimmune sera
on Westerns), immunization per se boosts production of many
other Ab and changes repertoire significantly. Thus comparing 
immune and preimmine might still lead to erroneous conclusions.

The best control is to use IgG purified from _the same serum_
after its depletion on the antigen column.

        - Dima


:Neal
:
:"If there are many universes, there will be one where there is a set of
:numbers suitable to life... we are in that one"
:
:Martin Rees, Astronomer Royal 
:
:Neal Melvin
:University of Calgary - Faculty of Medicine
:Department of Neuroscience - Neuroscience Research Group (NRG)
:Health Sciences Centre
:3330 Hospital Drive, NW
:Calgary, Alberta, Canada
:T2N 4N1
:
:e-mail: nrmelvin at ucalgary.ca
:Phone: (403) 220-4490 (lab)
:       (403) 220-7035 (office)
:       (403) 283-8731 (fax)
:        
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