I had some problems with my Westernblots lately. The SDS-Page went fine and the first antibody (PDGF-receptor alpha) I did looked really good. I striped the membrane and incubated it with aSMA afterwords. The results where really bad. I tried it twice and both times aSMA didn’t work after stripping. Have you ever had similar problems? I thought that maybe something went wrong with my wet blot, maybe not enough protein was transferred on the membrane to survive the stripping process or stripping itself wasn’t successful…
Sigma site has multiple protocols:
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/western-blotting/stripping-and-reprobing-western-blotting-membranes
Alternatively, you may be able to identify your colleagues at your place whom you could directly talk to and get help.
Are PDGF-receptor alpha and a-SMA antibodies from the same organism? If you can use different secondary antibodies (for example, rabbit for the first and mouse for the second) you can skip the stripping step and just incubate the membrane after first secondary antibody with 0.01% sodium azide to kill the HRP from the secondary antibody. Wash thoroughly (3 times with TBS-T/PBS-T, 10 min each) and incubate with the second primary antibody and then the correspondent secondary.
In this link you have suggested mild stripping buffers: https://www.researchgate.net/post/How_can_I_do_a_good_stripping_on_nitrocellulose_membranes_but_with_low_protein_loss
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