Immunoprecipitation with non-immune serum??

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Nov 1 10:17:42 EST 2000


In article <Pine.A41.4.10.10010290151380.58762-100000 at acs4.acs.ucalgary.ca>, Neal Robert Melvin <nrmelvin at ucalgary.ca> wrote:
:Hi, I'm starting to do IPs with a rabbit polyclonal antisera. I plan to
:use a non-immune rabbit antisera as a control, and in searching the
:literature, have noticed something strange... I've seen people that have
:used non-immune serum, and have a lane that is completely blank after
:detection with an anti-rabbit antibody... my question is: shouldn't the
:heavy/light chains of the non-immune serum be detected on a western blot
:when using an anti-rabbit secondary??

If there are IgG bands in sample there surely should be IgG in 
control. If there is not, it's either a fake or a failed experiment 
(in which case the "sample" is not to be trusted either).

Sometimes people go for trouble of crosslinking IgG used for IP to 
immobilized Protein A/G (in which case there won't be heavy chains 
on blot) or eluting bound protein under conditions that cause 
dissociation from ab but not ab from Protein A (in which case there 
won't be any IgG chains on blot).

        - Dima







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