Cells were lysed with ice-cold RIPA lysis buffer containing phosphatase-protease inhibitor cocktails (Beyotime Biotechnology, Shanghai, China). The concentration of protein was measured by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein lysates were subjected to SDS gel electrophoresis, immunoblotted with primary antibodies, and then the matched secondary antibodies. Western blot results were quantified by using the Image J software. Antibodies used in this study were listed in Supplementary Material .
Phosphatase protease inhibitor cocktails
Manufactured by Beyotime
Sourced in China
Phosphatase-protease inhibitor cocktails are a type of laboratory reagent used to inhibit the activity of phosphatases and proteases in biological samples. These cocktails contain a combination of chemical compounds that effectively inhibit the enzymatic activity of these biomolecules, which can help preserve the integrity and composition of the sample during experimental procedures.
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Lab products found in correlation
3 protocols using phosphatase protease inhibitor cocktails
1
Protein Extraction and Western Blot Analysis
2
Immunoprecipitation and Western Blot Analysis
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Cells were lysed with ice-cold IP lysis buffer containing phosphatase-protease inhibitor cocktails (Beyotime Biotechnology, Shanghai, China). After centrifugation at 12,000 rpm, the protein supernatant was incubated with primary antibody at 4°C overnight. The protein-antibody complex was pulled down with Protein A/G PLUS-Agarose Beads (Santa Cruz, Santa Cruz, CA, USA). Those beads were eluted with IP lysis buffer 4 times. Next, 45 μL 2× loading buffer was added to the agarose beads, 10 μL 5× loading buffer was added to the input, followed by boiling for 10 min. The IP protein was eluted for western blot analysis.
3
Immunoprecipitation Protein Extraction Protocol
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Cells were lysed with ice-cold RIPA lysis buffer containing phosphatase-protease inhibitor cocktails (Beyotime Biotechnology, Shanghai, China). After centrifugation at 12000 rpm for 20 min, the protein supernatant was mixed with the specific primary antibody and incubated at 4 °C overnight. The protein-antibody complex was pulled down with Protein A/G PLUS-Agarose beads (Santa Cruz, CA, USA). After 4 h, those beads were collected and then boiled with SDS-PAGE buffer to release the binding protein, and the immunoprecipitated protein was eluted for western blot analysis.
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