Western blots are usually set up with the antigen as the limitting factor, that
is, the antibodies (both primary and secondary) are in great excess relative to
the antigen so that the final bands seen on the blot are dependent only on the
amount of antigen in that band. The light band you described is likely to
already be saturated at the 1:3000 dilution. Increasing the antibody
concentration in such a situation will not make the band any darker, but will
increase the background of your blot, obscuring any faint bands. I would
suggest you go back to the 1:3000 dilution, and either increase the amount of
total protein you load on the gel or use a flourescent or radiolabelled
secondary antibody for more sensitive detection.
Dean Lee
Micro Dept
LLU
mbidle at lluvm.bitnet
