SQSTM1/p62 (D5E2) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in RIPA buffer containing 10% phosphatase inhibitor and 10% protease inhibitor cocktail.	- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. - Dilute primary Ab at 1:1000

Upstream tips
- Lyse cells in RIPA buffer containing 10% phosphatase inhibitor and 10% protease inhibitor cocktail.
Protocol tips
- Dilute primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. - Dilute primary Ab at 1:1000

Publication protocol

Treated cells were washed in ice-cold PBS and extracted for 30 min with a RIPA buffer containing 10% phosphatase inhibitor and 10% protease inhibitor cocktail (Sigma, St. Louis, MO). Lysates were cleared by centrifugation at 13,000 × g for 30 min and protein concentrations were determined using Bio-red protein assay. Equivalent amounts of whole cell extracts were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, MA, USA). The membranes were then probed with specific antibodies. Immune-complexes were detected using horseradish peroxidase conjugated with either anti-mouse or anti-rabbit followed by chemiluminescence detection (Perkin Elmer Cetus, Foster City, CA, USA). To demonstrate equal protein loading, blots were stripped and re-probed with a specific antibody recognizing GAPDH.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SQSTM1/p62 (D5E2) Rabbit mAb below.

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