I'm a beginner at western blotting, and I have hight background, However I don't know whether it comes from the primary or the secondary antibody. and I thought I could start over and incubate with the primary anti-body. Is it possible to do that after washing with TBS-T over night ?
If I understand you correctly, you are trying to use the same membrane again by washing away the primary and secondary antibodies? I would not recommend that. In theory you can wash away bound protein by using high concentration of surfactant, such as tween-20, however, in the meantime, you are eliminating both the antibodies and your bound target protein leaving the membrane empty.
You should try to use Stripping Buffer, block again and use your desired primary and secondary antibody
Hi, how is your background? Did you have a lot of aspecific bands? Did you have uniform or splotchy uneven background?
I agrred with the strip method but, regarding the backgorund think if you have to change the blocking reagent, the dilution of your antibodies or the ECL
I would recommend looking at your washing techniques, ensuring you are washing the membrane enough after each antibody is applied. Also consider your ECL and whether that is the best for your instrument and exposure requirements.
Another thing would be to address whether the blocking buffer you are using is best suited to your antibodies.
Unfortunately there are a lot of potential things that can cause high background, so you need to adjust one thing at a time.
You could try stripping your membrane but may also strip the protein away. You can check protein on your membrane using ponceau solution which washes away
Good luck
I would need more details to answer you properly, but If your problem is that your WB has more background than usual at first use, you could check you blocking stage. Primary antibodies are quite hard to optimise but companies have some specifications to use them. In case you wanted to use that membrane again, you should do a stripping before blocking it again.
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