The antibodies used in this study were: mouse and rabbit anti-NFBD1 (Abcam, London, UK); anti-ATM (phospho S1981) (Abcam); anti-DNA PKcs (phospho T2609) (Abcam); anti-phospho-H2AX (Ser139) (Cell Signaling Technology, Danvers, MA, USA); anti-phospho-histone H3 (Ser10) (Cell Signalling Technology); rabbit anti-Rad51 (Santa Cruz Biotechnology, Dallas, TX, USA). Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). Protein concentrations were determined using the BCA protein assay systems (Beyotime Institute of Biotechnology, Nantong, China). Proteins were fractionated in SDS–polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.
Bca protein assay systems
Manufactured by Beyotime
Sourced in China
The BCA protein assay systems from Beyotime are colorimetric assays used for the quantification of protein concentration. The assay works by the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the cuprous cation then chelating with bicinchoninic acid to produce a purple-colored complex that absorbs strongly at 562 nm. This method provides a simple, rapid, and accurate way to determine protein levels in solution.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Enhanced chemifluorescence detection system, by Cytiva (2 mentions)
Rabbit anti rad51, by Santa Cruz Biotechnology (1 mentions)
Anti phospho histone h3 ser10, by Cell Signaling Technology (1 mentions)
Anti phospho h2ax ser139, by Cell Signaling Technology (1 mentions)
Anti phospho atm s1981, by Abcam (1 mentions)
Anti nfbd1, by Abcam (1 mentions)
2 protocols using bca protein assay systems
1
DNA Damage Response Protein Analysis
2
Protein Extraction and Analysis
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Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the BCA protein assay systems (Beyotime Institute of Biotechnology). Proteins were fractionated in SDS–polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and Western blotting was performed using the appropriate antibody overnight at 4°C. This was followed by incubation with peroxidase-conjugated AffiniPure secondary antibodies for 1 h at 37°C. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA), and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences) at room temperature.
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