This is protocol that I suppose to follow
Buffer, 1 liter
15 g glycine
1 g SDS
10 ml Tween20
Adjust pH to 2.2
Bring volume up to 1 L with ultrapure water.
Membrane incubation
Use a volume that will cover the membrane. Incubate at room temperature for 5-10 minutes.
Discard buffer.
5-10 minutes fresh stripping buffer.
Discard buffer.
10 minutes PBS
10 minutes PBS
5 minutes TBST
5 minutes TBST
Ready for blocking stage
I am afraid you may need to repeat your Western. I don't see any chance that your blot will give trustworthy results. Stripping buffer incubation times are usually quite short. Overnight incubation even at 4°C is likely enough time work on your proteins.
I concur. Prolonged incubation may well have stripped away the protein from your lysate on the membrane distorting your result
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