I observe from a few experiments where I immunoblot with mab 4G10
(anti phosphotyrosine) and detect with GAM_HRP_ECL I get a diffuse
background that I don't see when I blot with other mouse monoclonals
or rabbit polyclonals. If I use 5% non fat dry milk dissolved in tris
buffered saline with 0.01% Tween 20 (TBS-T) as both a blocking agent
and a carrier for 4G10 I lose some of the signal and almost all of the
background. If I blot with the antibody in TBS-T I get improved signal
but the nasty looking background returns and is difficult to wash
1) Some component of the dry milk is competing away my signal.
2) Is that component phosphorylated tyrosine?
3) Would substituting BSA for milk help? Or is there some way of
preblocking or chemically treating the carrier?
Does anyone else have thoughts or experiences on the subject?