LC3B (D11) XP® Rabbit mAb

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	1:1000 dilution of Primary antibody

Protocol tips
1:1000 dilution of Primary antibody

Publication protocol

For western blot analysis, protein was extracted by radioimmunoprecipitation lysis (RIPA) buffer (Sigma-Aldrich, R0278) containing Halt Phosphatase Inhibitor cocktail (Thermo Fisher Scientific, 78420). Protein concentration was measured with a BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). NuPAGE® SDS PAGE Gel System (Thermo Fisher Scientific) was used for electrophoresis and western transfer, using 4 to 12% Bis-Tris gradient gel and PVDF membrane. The membranes were blocked and incubated with specific antibodies overnight at 4°C, washed 3 times for 5 min each with PBST (Thermo Scientific, 10010023; with 0.1% Tween 20; Bio-Rad, 161–0781) and incubated with an anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase (GE Healthcare Biosciences, NA9340 or NA9310). The membranes were washed again 3 times for 5 min with PBST, and antibodies were visualized after the addition of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095) and exposure to X-ray film. The primary antibodies used for the western analysis included: rabbit monoclonal anti-MAP1LC3B (1:1000; Cell Signaling Technology, 3868), mouse anti-SQSTM1 (1:2000; BD Biosciences, 610833), mouse anti-OSGIN1 (1:500; Sigma-Aldrich, SAB1407392) and mouse anti-ACTB/β actin (1:5000; Sigma-Aldrich, A2228). To quantify the western results, the films were scanned and analyzed with ImageJ software (http://imagej.nih.gov/ij/). ACTB staining was used for normalization.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3B (D11) XP® Rabbit mAb below.

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