Immunoprecipitation with non-immune serum??
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Nov 1 10:17:42 EST 2000
In article <Pine.A126.96.36.19910290151380.58762-100000 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).
More information about the Methods