HELP! Western blotting saga - (Sep/20/2006 )
We've tried pretty much everything, but nothing seems to be working. This week we got as far as knowing that the proteins have actually transferred, but when we apply the antibodies, nothing shows up, we've used the chemiluminescent kit as well as HRP staining, but nothing is showing.
Anyone have any ideas of what the problem may be?
can be anything; check the simpliest; some commercial ECL solutions lose effectivity even before date of expire; to your ECL solution add some H2O2 (about 1.5 µl per 5 ml ECL), you can double the amount of H2O2 but background may increase; if its not the ECL solution, primary or secondary Ab may be damaged; or check salt concentration or pH of TBS/PBS solution; check transfer efficiency by Ponceau S staining
do you have a positive control?
how did you determine the concentration of antibodies to use?
did you try to detect the primary antibody by dot blotting it?
Its difficult to point at a single problem.
confirm if transfer is efficient. then have a positive control to verify antibodies and ECL solutions.