LC3B (D11) XP® Rabbit mAb

Protocol tips

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	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

The cells were lysed in radioimmunoprecipitation assay buffer containing phosphatase inhibitor cocktail I (Sigma-Aldrich; Merck KGaA) and a protease inhibitor cocktail mini-tablet (Roche Diagnostics GmbH, Mannheim, Germany), followed by centrifugation at 8,000 × g at 4°C for 15 min. Protein concentration was determined by performing a bicinchoninic acid assay. An equal quantity of protein (50 µg) from each sample was separated on a 10, 12 or 15% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes. The membranes were probed with antibodies against P65 (rabbit IgG; cat. no. 4767; 1:1,000), p-P65 (rabbit IgG; cat. no. 8214; 1:1,000), GLUT-4 (rabbit IgG; cat. no. 2213; 1:1,000), LC3 (rabbit IgG; cat. no. 12513; 1:1,000), P62 (rabbit IgG; cat. no. 5114; 1:1,000) and β-actin (rabbit IgG, cat. no. 4970, 1:1,000; all Cell Signaling Technology, Inc.) at 4°C overnight. Following washing with PBS, a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; cat. no. 14708S; 1:2,000; Cell Signaling Technology, Inc.) was added to the membranes for 2 h at room temperature. The bound antibody was visualized using an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA).

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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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