how do you know your protein was on the membrane? did you stain with
ponceau or amido black or something like that?
if it was there, it should stay on. if you blocked your membrane with
protein (BSA, milk, ...), you have no way to find out if your e. coli
protein is there, cos there'll be protein all over the membrane.
maybe your antibody doesn't work.
try reprobing your blot with another antibody to see if you pick up a
signal. if you do, it means your blot is OK.
Good luck,
Rogier
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