When I started doing my Western Blots in February I had clean results, however, as time as progressed they have become messy (using HEK cells and also became messy in tissue). I haven't changed the protocol, and the primary antibody (pEphA2 invitrogen) I use is in glycerol stored at 4 degrees, and I have tried other pEphA2 invitrogen antibodies that we have and I am getting the same result. I have also had to increase the exposure time and use "Super ECL". I have consistently produced clean blots with other primary antibodies (e.g. pAKT, AKT, pEphA4, EphA4) even when cutting the blot in half. Any thoughts would be greatly appreciated!
some thoughts:
1. how much glycerol is there? maybe you can store your Antibody in -20 without freezing it.
2. did you run out of the old membrane and use a new batch?
3. what are you using for blocking? dried milk powder can go bad, esp in high humidity. how does it smell? cheesy?
4. are there other people in the lab who use the same reagents? how do their blots look like?
Thank you for your answer. Actually all of my other blots look fine. It is just the one with the pEphA2 antibody. Ive tried milk and BSA. The antibody is stored at -20 (as per info sheet provided with AB when purchased)
Have you tried giving the antibody a hard spin (max speed in a cooled microfuge for 5 min) and using the supernatant? Centrifugation will remove some aggregated protein that may have accumulated over time and often is responsible for the high background. Many antibodies seem to have a built-in mechanism of self-destruction that makes you buy it repeatedly. A fresh batch of the same antibody will possibly solve all your problems. I know this is not a satisfactory solution for a scientist, but it frequently is the only way to resolve it. Good Luck, Jürgen