SQSTM1 Antibody (D-3)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture	Dilute primary Ab at 1:1000 and incubate overnight at 4 °C.

Upstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4 °C.

Publication protocol

The cells were seeded into a 6 well plate with 2ml of cells per well. Following treatment, cells were collected and spun down. They were washed with PBS and lysed in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture (Roche) for 5 min and vortexed. The lysates were cleared by centrifugation at 12,000rpm for 10min. Protein samples were run on 8 or 15% polyacrylamide gels and transferred onto nitrocellulose membranes. Primary antibodies were incubated overnight at 4 °C. Each independent experiment was repeated three times. Protein amount was normalized with actin. The images were quantitated with ImageJ software (http://rsb.info.nih.gov/ij/).

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for SQSTM1 Antibody (D-3) below.

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