We received a limited amount of an antiserum antibody as a gift from another lab and are trying to avoid having to purify it. Our initial titration blot with blocking in 5% milk successfully detected the target protein, but encountered significant non-specific binding likely from high albumin-IgG in the serum.
Would using BSA for blocking decrease the non-specific binding, or could it exacerbate the issue due to additional albumin from the BSA? We have never performed westerns with unpurified antiserum antibodies before so any help or tips would be appreciated!
Edit: This is NOT a phospho-specific antibody
I think nonfat dry milk milk and BSA (1-5% each) work equally well as blocking agents for Western blots. Milk is much less expensive than BSA. Use the name brand (Carnation) if it's available.
I'm not sure what you mean by "additional albumin from the BSA." BSA is albumin. The blocking agent in milk is casein. You can also by protein-free blocking agents, which are expensive but useful when you are using strepatividin-based detection, since biological blocking agents can contain biotin.
A good way to remove unwanted immunoglobulins when you only have a little antiserum is to affinity purify on a micro scale. Preincubate a small amount of the antiserum with a sample containing the unwanted antigens but not the desired antigen, if such a sample is available or can be prepared.
I always use milk 1% (w/v) for 1º Ab, and 0.1% milk with the 2º when doing WB. The buffer we use is: PBS-Tween20(0.05% (v/v)-defated-milk (x%). Between antibodies is recommended to do 3 washes with PBS-Tween for 5 minutes each one, and previously to the 1º Ab you must do a bloking step for avoiding non-specific binding of the antibodies.
This blocking step is usually 1 hour RT or O.N. 4ºC, with PBS-Tween20(0.05%)-defated milk 5% (w/v)