i'm having problems determining antibody specificity.

James jwhite at systemsbiology.org
Mon Aug 25 18:29:07 EST 2003

I have gone to the effort of creating a monoclonal antibody.  On a
western several bands appear.  To test which of these bands is
specific I conducted a blocking assay using the recombinant protein
towards which the antibody was produced.  I incubate the antibody
(~3.0ug) with varying amounts of protein (3ug, 6ug, 10ug, 25ug, and
50ug)in 200ul of 1x PBS first for two hours at 37C then at 4C
overnight to 24hrs.  I then spin the immune complexes out at the
highest speed in a microfuge for 15min.  I then add the supernatant to
2.5ml of blocking buffer and conduct westerns with the mixes.  My
problem is that I obtain very high background in the blocked samples,
so much so that the whole blot turns black during the exposure time it
takes to see the bands in the control (everthing same except no
recombinant protein added).  A very short exposure time results in
bands showing up, however the incorrect bands, with high background. 
I've done the same with another antibody that I've made.  I've had no
problems with this one.  Can anyone help me?  Thanks alot.


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