Immunoprecipitation with non-immune serum??

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Sun Oct 29 16:00:21 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?? Doesn't seeing absolutely
nothing in
> the non-immune serum lane indicate that the rabbit antibodies didn't
bind
> to the protein A in the first place??
>

Well, that does sound reasonable. Two possible reasons for not seeing
anything: (1) They are only showing part of the gel that doesn't have
IgG (e.g., > 60 kDa) or (2) they are probing the western with a
different (non-rabbit) Ab. For example, you could IP with rabbit, probe
the blot with mouse Ab, and depending on the quality and specificity of
the anti-mouse secondary, you may not see the rabbit IgG chains light
up.

In the papers that you are referring to: do the Ab chains show up in
the other lanes?

Nick

--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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