So, we're having problems with immunoblot with AntiCol1A1. We're having a big background and we can barely see some bands we have already made with the same samples and, despite having some background, we could see the bands, but as one of our gels broke, the transfer was not so good and we hadn't united bands in the membrane. We repeated and we had a big background, we let 2hours to lose a bit of chemiluminescence signal, but we lose all the signal in one membrane and the other had only some parts, but it was the background signal.
We're doing immunoblot with PVDF membranes, this is the protocol:
-Blocking with 10% milk powder + 1xTBS-T, O/N at 4ºC
-Wash with 1xTBS (2x5')
-Incubation with first Antibody Goat AntiCol1A1 (sc8783) 1:1000 1h RT
-Wash with 1xTBS-T (15' + 2x10')
-Incubation with secondary Antibody AntiGoat-HRP (A5420)
-Wash with 1xTBS-T (15' + 10') + 1xTBS (2x5')
-ECL Revelation
With the other membranes, where we could see the bands, we dried the membrane after transferring them and store them at 4ºC in the weekend, next monday we reactivated them again with methanol and let them O/N with blocking solution (10% milk powder + 1xTBS-T) at 4ºC.
What could be the problem?
In my experience letting the membrane dry causes high background signal. If you have to wait before developing, just leave it in TBST buffer or PBS next time. I don't like to use milk in the blocking solution, since it can also cause high background. I would use either BSA or serum from whatever species the secondary antibody is, probably donkey, I suspect. Did your gel brake during the transfer? What type of transfer are you doing? Maybe you lost the protein during the transfer. If you still have the membrane you can use a protein dye to see if you have any bands left.
Yes, I agree, try to switch to 3% BSA solution, also do not block the membrane overnight. 1 h at RT is enough, whereas incubation with the primary antibody may be much longer (try to leave overnight at RT). Try to wash 4x5min with TBST after each step. Make sure the Antobody solution does not contain sodium azide, since it interferes with HRP-tagged Ab. Make sure your transfer time is optimized. Try to use Nitrocellulose 0.45 uM pore size, it's perfect for the molecular weight of your protein of interest. If nothing works out, I would question the quality of the antibodies, both primary and secondary.
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