Beclin 1 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Dilute Ab at 1:10000 in blocking solution. - incubate primary antibodies overnight at 4°C.

Protocol tips
- Dilute Ab at 1:10000 in blocking solution. - incubate primary antibodies overnight at 4°C.

Publication protocol

Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer containing freshly added 0.01% protease inhibitor for 30 min. After the lysed cells were incubated on ice for 10 min, they were centrifuged at 12000 × g for 10 min at 4°C, and the supernatants were collected. Approximately 50 μg of total protein was denatured for 10 min at 95°C, separated on a 10–15% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature and then incubated in primary antibodies overnight at 4°C. The primary antibodies were detected by incubating the membranes in horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, and the signals were visualized using a SuperEnhanced Chemiluminescence Detection Kit. Details of the primary and secondary antibodies are presented in S-Table 3. Each assay was performed in triplicate.

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Manufacturer protocol

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