Western blots were carried out on 40 µg of protein extracted with NTEN (20 mM Tris at pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) plus protease and phosphatase inhibitors. All of the antibodies were incubated overnight at 4°C in 2% milk. The antibodies used were rabbit anti-Rad50 polyclonal, rabbit anti-Nbs1 polyclonal, and rabbit anti-Mre11 polyclonal previously described (49 (link)), ATM (Cell Signaling), RPA32 pSer4/Ser8 (Bethyl Laboratories), Chk1 (C9358, Sigma), Chk1 pSer345 (Cell Signaling), AID (50 (link))), GAPDH (6C5, Millipore) and Smc1 (Cell Signaling).
Protocol detail
Western Blot Analysis of DNA Repair Proteins
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Antibodies
Edta
Gapdh
Inhibitors
Milk
Nacl
Np 40
Phosphatase
Protease
Protein
Rabbit
Rad50
Tris
Western blots
Corresponding Organization : Kettering University
Other organizations : Memorial Sloan Kettering Cancer Center, University of Pennsylvania, Children's Hospital of Philadelphia, Cancer Research Institute
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Variable analysis
independent variables
- 40 μg of protein extracted with NTEN (20 mM Tris at pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) plus protease and phosphatase inhibitors
- Rad50 protein levels
- Nbs1 protein levels
- Mre11 protein levels
- ATM protein levels
- RPA32 pSer4/Ser8 levels
- Chk1 protein levels
- Chk1 pSer345 levels
- AID protein levels
- GAPDH protein levels
- Smc1 protein levels
- Antibody incubation conditions (overnight at 4°C in 2% milk)
- Rabbit anti-Rad50 polyclonal, rabbit anti-Nbs1 polyclonal, and rabbit anti-Mre11 polyclonal antibodies previously described (49)
- Not explicitly mentioned
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