I am using polyclonal antisera (rabbit) for western-blot detection. The
antibodies were raised against a MBP-fusion protein and are not purified.
Although there are some unspecific cross-reactions, a clear detection of the
antigen is possible.
My problem is, that the signals after detection with HRP-conjugate are not
always of the same strength, although the blotting itself (controlled by
Ponceau-staining) seems to work well. Can this be due to an inhomogeneous
distribution of specific antibodies in the serum (I use a final dilution of
1:3000 in a total volume of 20 ml buffer), or is there something else going
wrong?
Thanks for your help!
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