The Western Blot technique for identification of specific proteins may have several problems that need to be resolved. One of these is a diffuse or nonspecific band.
Situation: diffuse or nonspecific band
Problem
Possible Solution
antibody concentration too high
reduce the concentration of the primary and other antibodies
too much protein loaded onto the gel
reduce the volume of sample loaded onto the gel
signal from chemiluminescent substrate too strong
try one or more of the solutions listed below
Possible solutions for a strong signal:
(1) reduce the film exposure time
(2) reduce the concentration of the substrate
(3) shorten the incubation time of the membrane with the substrate
(4) completely remove substrate after the incubation period
(5) decrease the concentration of antibodies, particularly conjugated antibodies
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