Patchy Western Blots (particularly anti-phosphotyrosine blots)

[dicleme4]

Hi all,

I have been having occasional problems with my western blotting where I get large patches with no signal at all and then lots of background everywhere else (attached is an example of a anti-phosphotyrosine blot).

I don't believe it's a transfer problem as I have done a fast green stain and I can see bands of protein on the entire membrane.

I don't think it is an ECL problem as I have reapplied ECL and the same patchy patterns appear.

I don't think it is a problem of the distribution of secondary antibody because if I rinse the membrane with TBST and then incubate again with secondary, followed by ECL the same patterns appear.

I feel it could be a primary antibody problem (maybe the primary antibody is just not getting to those patchy parts of the membrane?) but if I rinse off the ECL, reapply primary, secondary, and ECL, I still see the same patterns.


As this doesn't happen to me all the time, only sometimes, I feel I am doing something technically wrong. No one in my lab has seen this problem, and someone has watched me perform the western blot and can't seem to figure out what my problem is.

I'm hoping someone has seen something like this before and can give suggestions.

Thanks!