Dear all, I have an interesting problem. I performed a WB but the initial primary antibody incubation produced weak bands after 20 minute exposure. So I reincubated with the same antibody solution the membranes. I found that the signal improved, although to my surprise the weaker bands (2 out of 12) seen in the first exposure wore now one of the strongest. So the differences between the samples changed upon the second incubation. Do you have any ideal what could be the explanation?
If you are using ECL, the reaction is known to inactivate the HRP, particularly if you have a strong signal.
Therefore, it is possible that the HRP-conjugated antibody from the 1st incubation is still there, but the HRP is not (fully) active anymore at the main band.
On the other hand, the bands that showed weak signal in the 1st ECL incubation might have survived, and might gave relatively stronger signal in the second ECL incubation.
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