LC3B Antibody

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	Dilute Primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute Primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

The brain homogenate collected from SD rats and the cultured neurons were lysed in RIPA lysis buffer (P0013; Beyotime, Shanghai, China). We centrifuged the samples at 12,500 × g for 15 min at 4°C, collected the supernatants, and determined the protein concentration of the supernatants by standard BCA (P0012; Beyotime, Shanghai, China) method. Then, the samples (100 μg/lane) were loaded onto a 15% SDS polyacrylamide gel, separated, and electrophoretically transferred to a polyvinylidene difluoride membrane (IPVH00010; Millipore, Billerica, MA, United States). The membrane was blocked in 5% bovine serum albumin (BSA, BIOSHARP, Hefei, AH, China) for 2 h at room temperature. Subsequently, the membrane was further incubated with the primary antibody overnight at 4°C and then incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The membrane was revealed with the enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) and the relative quantities of proteins were analyzed with Image J software.

To test the level of p-PEBP1 by western blot easily, we enriched PEBP1 from total proteins by immunoprecipitation using PEBP1 antibody before electrophoresis.

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