Co-IP was performed as described previously (Sharma et al., 2019a (link)). Cells were washed with 1× PBS and cell lysates prepared in cold triton lysis buffer (1% Triton X-100; 40 mM HEPES, pH 7.5; 120 mM sodium chloride; 1 mM ethylene diamine-tetraacetic acid; 1 mM phenyl methylsulfonyl fluoride; 10 mM sodium pyrophosphate; 1 μg/ml each of cymostatin, leupeptin, and pepstatin; 10 μg/ml each of aprotinin and benzamidine; 2 μg/ml antipain; 1 mM sodium orthovanadate; 50 mM sodium fluoride) supplemented with 0.2% empigen for anti-K19 IP, anti-GSK3β IP, anti-myc IP or anti-GFP IP. Cell lysates were centrifuged to remove cell debris, and protein concentration was determined using the Bio-Rad protein assay with BSA as standard. Aliquots of cell lysate were then incubated with the indicated antibody or IgG control, and immune complexes were captured using either Protein G Sepharose (GE Healthcare) or GFP-Trap beads from Chromotek (Islandia, NY).
Protein assay with bsa
Manufactured by Bio-Rad
The Protein assay with BSA is a laboratory product designed for the quantitative determination of protein concentration. It utilizes bovine serum albumin (BSA) as a standard for the measurement. The assay provides a reliable and consistent method for protein quantification in various sample types.
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2 protocols using protein assay with bsa
1
Co-Immunoprecipitation Assay Protocol
2
Co-immunoprecipitation Protocol for Protein Analysis
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Co-immunoprecipitation was performed as described previously (Sharma et al., 2019a) . Cells were washed with 1X PBS and cell lysates prepared in cold triton lysis buffer (1% Triton X-100; 40 mM HEPES (pH 7.5); 120 mM sodium chloride; 1 mM ethylene diamine-tetraacetic acid; 1 mM phenyl methylsulfonyl fluoride; 10 mM sodium pyrophosphate; 1 μg/ml each of cymostatin, leupeptin, and pepstatin; 10 μg/ml each of aprotinin and benzamidine; 2 μg/ml antipain; 1 mM sodium orthovanadate; 50 mM sodium fluoride) supplemented with 0.2% empigen for anti-K19 IP, anti-GSK3 IP, anti-myc IP or anti-GFP IP. Cell lysates were centrifuged to remove cell debris, and protein concentration was determined using the Bio-Rad Protein Assay with BSA as standard. Aliquots of cell lysate were then incubated with the indicated antibody or IgG control, and immune complexes were captured using either Protein G Sepharose (GE Healthcare) or GFP-Trap beads were from Chromotek (Islandia, NY).
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