Beclin-1 (D40C5) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in RIPA buffer with protein inhibitor	Dilute primary Ab at 1:1000 and incubate overnight at 4°C with gentle shaking

Upstream tips
Lyse cells in RIPA buffer with protein inhibitor
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C with gentle shaking

Publication protocol

The cells were harvested and washed twice with ice-cold PBS buffer in 1.5 ml EP tube, centrifugedat 3000 g for 5 min. The supernatant was removed and RIPA buffer with protein inhibitor was added. The mixture was placed in ice for 30 min. Lysates were centrifuged at 12,000 g for 30 min, and the protein in the supernatant was denatured with lysis buffer for 5 min at 100°C and then subjected to SDS-PAGE gels. The gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, CA); the membranes were blocked with 5% nonfat milk in TBST for 1 h and incubated with a primary antibody overnight at 4°C. The primary antibodies used were rabbit anti-Beclin-1 (1:1000, CST, D40C5), rabbit anti-LC3 B (1:1000, CST, 12513S), rabbit anti-bcl-2 (1:1000, 2870 s) and mouse anti-β actin (1:5000, sigma). Bound secondary antibodies were visualized using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology) with ChemDoc XRS with Quantity One software (BioRad, Hercules, CA).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Beclin-1 (D40C5) Rabbit mAb below.

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