Cells were cultured in 3.5 cm diameter plates (80–90% confluence), washed by PBS buffer, and lysed for 15 min on ice using a RIPA buffer (#C2978, Sigma, St. Louis, MO, USA) containing an anti-protease mix (#PI78415, Thermo Scientific, Waltham, MA, USA). Protein concentration was measured by BCA assay (#23225, Thermo Scientific, Waltham, MA, USA). Equal amounts of proteins were subjected to SDS-PAGE and immunoblotting as described previously [23 (link)].
Anti protease mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-protease mix is a solution containing a combination of protease inhibitors. It is designed to protect proteins from degradation by proteolytic enzymes during sample preparation and analysis.
Automatically generated - may contain errors
2 protocols using anti protease mix
1
Cell Lysis and Protein Quantification
2
Quantitative Western Blot Analysis
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Cells were cultured in plates 3.5 cm in diameter (80–90% confluence), washed by PBS buffer, and lysed for 15 min on ice using radioimmunoprecipitation assay buffer (C2978, Sigma–Aldrich, St. Louis, MO) containing an antiprotease mix (PI78415, Thermo Scientific, Waltham, MA). Protein concentration was measured by bicinchoninic acid (23225, Thermo Scientific). To alleviate the effect of membrane overexposure, serial dilution of cell lysates starting from a 50 μg sample over at least 10 protein dilutions was applied to generate a standard curve of band density for each protein target. Quantification of band density of each target was achieved by ImageJ software. To assure that the densitometric data for all of the target proteins will be within the linear quantitative range, 15 μg of proteins were subjected to SDS-PAGE and immunoblotting.
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