All primary monoclonal antibodies (mAb) were purchased from commercial vendors: pS343-Nbs1 (rabbit mAb, Abcam), γH2AX (mouse mAb, Millipore), and Rad51 (rabbit mAb, Epitomics; mouse mAb, Novus Biologicals). Certificates of Analysis were provided for all antibodies. Antibodies were custom-conjugated to fluorescent dyes or haptens: anti-γH2AX-biotin (Millipore), anti-γH2AX to Alexa Fluor 790, anti-pS343-Nbs1 to Digoxigenin (DIG) and anti-Rad51 to Dinitrophenol (DNP) (Molecular Probes, Inc.). New and previously qualified lots of antibodies were compared side-by-side. Validation details can be found in the Supplemental Methods and Supplemental Figs. S1 and S2 , and in LoRusso, et al. for pS343-Nbs1 (6 (link)). Cell cycle and apoptosis antibodies used were: p21 Waf1/Cip1 (12D1), cyclin B1 (D5C10), p-histone H3-Ser 10 (rabbit mAb, Cell Signaling Technologies, Danvers, MA), cyclin B1-Alexa-647 (rabbit mAb, [EPR17060] Abcam), geminin (mouse mAb, Abcam), and cleaved caspase-3 rabbit antibody (R&D Systems).
Protocol detail
Antibody Validation for Cell Cycle Analysis
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Antibodies
Antibody
Apoptosis
Biologicals
Biotin
Caspase 3
Cell cycle
Cyclin b1
Digoxigenin
Dinitrophenol
Figs
Fluorescent dyes
Geminin
Haptens
Histone h3
Molecular probes
Monoclonal antibodies
Mouse
Novus
Rabbit
Waf1 cip1
Corresponding Organization : Frederick National Laboratory for Cancer Research
Other organizations : Center for Cancer Research
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Variable analysis
independent variables
- PS343-Nbs1 (rabbit mAb, Abcam)
- γH2AX (mouse mAb, Millipore)
- Rad51 (rabbit mAb, Epitomics; mouse mAb, Novus Biologicals)
- Cell cycle and apoptosis antibodies: p21 Waf1/Cip1 (12D1), cyclin B1 (D5C10), p-histone H3-Ser 10 (rabbit mAb, Cell Signaling Technologies, Danvers, MA), cyclin B1-Alexa-647 (rabbit mAb, [EPR17060] Abcam), geminin (mouse mAb, Abcam), and cleaved caspase-3 rabbit antibody (R&D Systems)
- New and previously qualified lots of antibodies were compared side-by-side
- Certificates of Analysis were provided for all antibodies
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