Western not working - (Jul/03/2013 )
We've got some serious and weird problem with our western blots. All our western blots have stopped working. It's not the gel or transfer as we get good staining on the membrane by ponceau. It's also happening for all our primary antibodies, not one, so it's unlikely to be that. We've got a new secondary HRP and new ECL reagent. The westerns give no signal on film - then give a good signal and then give no signal - all with no warning or any reason I can think of. Has anyone got any ideas?
Maybe the HRP of your secondary antibody is knackered? A quick way to test this is to prepare 1ml ECL in a 1.5ml tube, go to the dark room and add a bit of Ab (1µl neat, or a low dilution), You should see light with the naked eye (remember to be in the dark). If this doesn't work something is wrong either with the HRP or the ECL.
Hope this helps... is a very quick test and fun
edit: Also (though less fun), check the pH of your wash and incubation buffers as this might affect the antibodies, and I assume this is common for all your westerns not working.
Also check that your fridge is still running - having antibodies at room temp for a while will stop them working quite effectively.
Cheers guys - already checked the HRP - works fine on some TMB and the fridge is fine. Buffers is a good point - I'll try that. Not sure if it could be as their all commercially bought in, but I'll grab for anything at the moment!
make sure there's no azide with the secondary.