I'm trying to detect the expression of cleaved caspase3 in mouse brain by western blot. Basically, my protocol is as followings:
1, lysis buffer: RIPA (ThermoFisher Science)
2, 50ug protein was loaded
3, 15% SDS-GAGE polyacrylamide gel
4, PVDF membrane
5, transfer: ice cold, 10%mathonal, 100V * 1hour
6, 5% non-milk blocking
7, primary antibody: anti-caspase 3, Bioscience, diluted to 1:500
But the cleaved caspase3 can't be detected.
By the way, LC3I has never been detected using the same protocol.
Can anyone give me any suggestion? Thank you!
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