Cells were collected after 5% trypsinization of the culture plate, followed by centrifuging at 12,000 rpm at 4 °C for 10 min. Cell pellets were resuspended in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor sodium orthovanadate (Santa Cruz Biotechnology Inc., Dallas, TX, USA) on ice for 30 min with vortex every 10 min. Protein concentrations were measured using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). An amount of 20 µg proteins from each sample were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (PerkinElmer, Waltham, MA, USA). Membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich). To detect target proteins, membranes were probed with specific primary antibodies overnight. The next day, membranes were washed for 10 min with TBS-T 3 times, followed by incubation with anti-rabbit or anti-mouse secondary antibodies. Protein bands were detected using chemiluminescence reagents (Millipore, Billerica, MA, USA).
Phosphatase inhibitor sodium orthovanadate
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sodium orthovanadate is a phosphatase inhibitor, commonly used in biochemical and cell biology research. It inhibits the activity of various protein tyrosine phosphatases and serine/threonine phosphatases.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by PerkinElmer (2 mentions)
Chemiluminescence reagent, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Geldoc go imaging system, by Bio-Rad (1 mentions)
2 protocols using phosphatase inhibitor sodium orthovanadate
1
Western Blot Protocol for Protein Detection
2
Western Blot Protein Analysis Protocol
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Cells were harvested by 0.05% trypsin from the culture plate and centrifuged at 12,000 rpm 4°C for 2 min. Cell pellets were re-suspended in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor sodium orthovanadate (Santa Cruz Biotechnology). The suspension was slowly shaken on ice for 30 min, with a vortex every 10 min. Protein concentrations were measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Proteins (10 μg) from each sample were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (PerkinElmer, Waltham, MA). Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich). Membranes were probed with specific primary antibodies overnight at 4°C to detect target proteins. On the second day, membranes were washed for 10 min with TBS-T three times, followed by incubation with anti-rabbit or anti-mouse secondary antibodies for 2 h. Then membranes were washed 10 min × 3 times with TBS-T. Protein bands were detected using chemiluminescence reagents (Millipore, Billerica, MA) in GelDoc Go Imaging System (Bio-Rad).
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