http://www.all-about-films.com/film-western-blot/
what are the common errors for not getting protein bands when i do film, after doing western blot and probing?
i probe with antibody primer which is diluted 3000 times. secondary antibody (HRP) is also diluted 3000 times. i detect the bands using ECL, then i put the membrane in the cassette. i put the film in developer solution for 20 seconds and fixer solution for 15 seconds.
many factors are responsible for this negative result.
1: antibody is not recognizing the protein ( so check for + control). Check whether your ab recognizes only native protein, not denatured as happens after SDS-PAGE.
2: Ab dilution (primary ab)
3: I would not consider ECL is bad but if you have + control then this can be rulled out.
good luck
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