Possible Cause & Solution
a. Too much protein per lane.
Titrate down the amount of protein loaded per lane.
b. Insufficient blocking of non-specific binding.
Adjust blocking conditions.
Include blocking agent in the antibody buffers as well.
c. The primary antibody concentration may be too high
d. Titrate the antibody to find the optimal concentration.
e. The secondary antibody may be binding non-specifically. Blot with the secondary antibody alone. If bands develop, choose an alternate secondary antibody.
f. Incubation temperature may be too high. Incubate blot at 4°C.
g. Cross-reaction between blocking agent and primary or secondary antibody.
Add a mild detergent, e.g. Tween®-20, to the incubation and washing buffers for phosphoprotein specific antibodies. Use BSA as a blocking reagent instead of milk. Milk contains casein, a phosphoprotein, and your antibody may be cross-reacting with the casein.
h. Washing of unbound antibodies may be insufficient.
Increase the number of washes.
i. The membrane may give high background.
Nitrocellulose membrane may give less background than PVDF.
j. The membrane has dried out.
Avoid drying out the membrane during processing and incubation.