After treating my membrane with the primary antibody, the destaining buffer was accidentally used instead of tbst buffer. Immediately after use (about 3 seconds later) it was replaced with tbst buffer (10 minutes, 4 times wash), will it affect the antibody signal? The composition of the destaining buffer is acetic acid, methanol, and 3dw.
I would expect exposure to destain solution (methanol/acetic acid), even for a short time, to negatively effect the interaction of the primary antibody with the antigen for 2 reasons: the organic solvent will have a denaturing effect on the antibody, and the acidity will interfere with antibody binding (low pH is used to elute antibodies from antigen affinity columns and vice versa). The brief duration of the exposure (3 seconds) may mitigate these effects to some extent, however.
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