I developed a western blot to see phospho-ABCA1 254kDa (good signal). Then I striped the membrane and label with anti-ABCA1 and didn't see ABCA1. I marked with Ponceau red and the nitrocellulose membrane (0.45 um) was completely white. I lost the proteins. I have never seen this before. I used the stripping protocol repeatedly in the same membrane and I have never had any problems. Maybe the problem is the transfer protocol. I did the transfer for 1h 30 min at 100mA (wet, cold).
Check the following...
1. your total ABCA1antibody is from rabbit or mouse and you had used appropriate secondary antibody.
2. confirm you had methanol in the transfer buffer which helps stronger binding of proteins with membrane.
3. Stripping usually leads to little loss of protein so reduce the time of stripping.
4. If you use beta-mercaptoethanol, and if you havent washed properly after stripping, it can target the disulphide bonds in the antibody (total ABCA1) so that the antibody cannot bind to ABCA1..but since you say the membrane looked white after ponceauS staining, it appears to me that you had done over stripping.
5. You had got phospho antibody signals so, it is not transfer problem.
6. Do a fresh blot, do phospho antibody probing, and then wash 3 times with distilled water for 5 mins each, then add 0.2M NaOH just to cover the membranes and incubate at 56 degree C for 3-4 minutes with shaking (do not exceed this time limit) and immediately discard NaOH, and quickly rince with distilled water 3-4 times and wash with distilled water twice (5 mins each), and wash with PBST/TBST for 5mins once. NaOH can also damage the antibodies you will be using later, thatswhy I recommend several water washes and PBST/TBST wash. Then block the membrane for 15 mins (this is good enough because this is blocking for second time), and proceed for the ABCA1 primary antibody over night incubation at 4 degree C. You should get proper bands after secondary antibody+ECL. Good luck.
Thank you for your answer. I can't incubate at 56 degree C with shaking. Can I incubate with 0.2M NaOH a room temperature more time?
I think if you use a water bath for 56 degree (without shaking) should be fine. I dont know how long it takes in room temperature. I havent done that.
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