Purified Mouse Anti-Beclin Clone 20/Beclin (RUO)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 0.125 mol/L Tris, pH 6.8, 4% SDS, 20% glycero, 10% 2 mercaptoethanol	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in buffer containing 0.125 mol/L Tris, pH 6.8, 4% SDS, 20% glycero, 10% 2 mercaptoethanol
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Publication protocol

Cells were lysed using cell lysis buffer (0.125 mol/L Tris, pH 6.8, 4% SDS [Sigma-Aldrich, L3771], 20% glycerol (Sigma-Aldrich, G5516), 10% 2-mercaptoethanol [Sigma-Aldrich, M625]. CBX protein assay kit (G-biosciences, 786-12X) was used to determine the protein concentration per the manufacturer's protocol. Cell lysate (30 to 60 µg) was electrophoresed in 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes at RT. Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, sc-2325) in 0.05% Tween 20 (Sigma-Aldrich, P9416) in 1x PBS. The membranes were incubated with primary antibodies for BECN1 (1:1000; BD Biosceinces, 612113), ATG3 (1:1000; Cell Signaling Technology, 3415P), LC3B/MAP1LC3B (1:1000; Novus Biologicals, NB100-2220) and TUBB/β tubulin (1:4000; Thermo, RB-9249-P1) in PBS-Tween 20 having 1% BSA and incubated overnight at 4°C. The blots were subsequently incubated with the secondary antibody conjugated to horseradish peroxidase (Abcam, ab6721)at 1:3000 dilutions in 0.05% PBS-Tween 20 for 1 h at RT. Protein bands were observed on X-ray film using femto LUCENT™ PLUS-HRP Chemiluminiscent reagent kit (G-biosciences, 786-003).

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Manufacturer protocol

Download the product protocol from BD Biosciences for Purified Mouse Anti-Beclin Clone 20/Beclin (RUO) below.

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