I've repeated this western blot multiple times, I've used three batches of freshly extracted samples. The PVDF membrane was always cold and opened right before the blocking, I've touched it only with tweezers on one edge. For the blocking I've used a 5% NFDM in 1X T-TBS and sodium azide and the membrane was incubate for 1h at room temperature. For the antibodies, I've initially used some aliquotes already tested and then, I've also prepared a new aliquotes. Every time I've washed the membrane 5 times (3x of 10 min and 2 of 5 min). The development was carried out using normal ECL (1:1 dilution) with incubation time of 3 minutes. Despite that, I continue to have no protein signal (the protein is expressed) and a lot of dirty on my membrane, but can't figure out what's the problem, and I'm running out of motivation since my prof continue to mock me for those results
I can see you have some signals but very high background
Just some questions for you
1. Which buffer did you dilute your primary and secondary antibodies in?
2. At what concentration did you dilute them? this is important as higher concentration may lead to higher backgrounds
For blocking, I usually use 5% BSA in PBS-T(Tween-20) or 5% milk in PBST and also dilute primary antibody (overnight in cold room) in same buffer and I dilute secondary antibody in 1% BSA in PBST or in 1% milk in PBST (room temperature). I use BSA and not milk if I am probing for a phosphoprotein as milk contains phosphoproteins which may cause similar issues like you have encountered
If you are reusing your antibodies, try to prepare new antibodies and test run. Also, make sure your wash buffers are freshly prepared if necessary.
Regards
Ayodeji Abijo Thanks for your answer. My primary and secondary antibodies are both diluted in 5% NFDM in TBS-T, and the concentration of primary antibody depends on their datasheet (1:1000 or 1:2000 usually), while the secondary one is 1:5000 (usually they are powerful). The blocking occurred for 1h RT using the same 5% NFDM, incubation with primary antibody was ON 4°C on slow oscillation, while the secondary antibody was incubated for 1h at RT
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