Anti-Beclin 1 (Human) pAb

Protocol tips

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

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

Publication protocol

Cells were washed with phosphate-buffered saline (PBS; Sigma-Aldrich, D8662), scraped in ice-cold PBS and centrifuged at 500 g for 5 min. The cell pellets were resuspended directly in reducing sample buffer (Laemmli; 60 mM Tris-HCl, pH 6.8, 2% SDS (Gibco, 15553-035), 100 mM dithiothreitol and 0.01% bromophenol blue) in the presence of a complete EDTA-free protease inhibitors cocktail (Roche Diagnostics, 04693159001). Genomic DNA was sheared by passage through a narrow-gauge syringe in order to reduce viscosity and resulting total protein extracts were then heated at 95°C for 4 min. Proteins were separated on a SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Dutscher 44087). Blots were blocked for 1 h with Tris-buffered saline (50 mM Tris.HCl, pH 7.4, 150 mM NaCl)-0.05% Tween 20 (TBS-T) supplemented with 5% nonfat milk and incubated overnight at 4°C with primary antibody. Filters were then washed in TBS-T, incubated for 45 min at room temperature with appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, sc-2004 and sc-2005) and washed again prior to detection of signal with ECL plus chemilumiscent detection kit (Thermo Scientific, 80196). Primary antibodies used in this study were rabbit polyclonal anti-LC3 (L8918), rabbit polyclonal anti-ATG7 (A2856), mouse monoclonal anti-ACTB/β-actin (clone AC-15, A1978) and mouse monoclonal anti-TUBB/β-tubulin (clone TUB 2.1, T4026) antibodies from Sigma-Aldrich and rabbit polyclonal anti-BECN1/beclin-1 antibodies from MBL International Corporation (CliniSciences, PD017).

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