For native RIP, MDA-MB-231 extract was incubated with 10 μg of anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) and then with Protein A Sepharose beads. After a total of three washes in RIP buffer, beads were boiled in SDS buffer for Western blot, or resuspended in TRIzol reagent for real-time RT-PCR. UV-Crosslink RIP (CLIP) was performed as described34 (link)–36 (link). Briefly, UV-irradiated MDA-MB-231 cells were lysed in RSB-Triton buffer, incubated with anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST), and then precipitated with Protein A Sepharose beads. Beads were then extracted for Western blot or real-time RT-PCR.
Protocol detail
Investigating DNA Repair Protein Interactions
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Anti dna
Antibody
Buffer
Clone
Dna pkcs
Ku70
Mda mb 231 cells
Pkcs
Protein a sepharose
Real time pcr
Trizol
Western blot
Corresponding Organization :
Other organizations : University of Pennsylvania, The Wistar Institute, UPMC Hillman Cancer Center
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Variable analysis
independent variables
- Anti-Ku70 antibody (Cat No:MA5-13110, Clone No:N3H10, Thermo)
- Anti-Ku80 antibody (Cat No:MA5-12933, Clone No:111, Thermo)
- Anti-DNA-PKcs antibody (Cat No: MA5-13404, Thermo)
- Control IgG (Cat No:5415S, CST)
- Protein expression (measured by Western blot)
- RNA expression (measured by real-time RT-PCR)
- MDA-MB-231 cell line
- Protein A Sepharose beads
- RIP buffer
- TRIzol reagent
- UV-irradiation of MDA-MB-231 cells (for CLIP)
- RSB-Triton buffer (for CLIP)
- Anti-Ku70 antibody (Cat No:MA5-13110, Clone No:N3H10, Thermo)
- Anti-Ku80 antibody (Cat No:MA5-12933, Clone No:111, Thermo)
- Anti-DNA-PKcs antibody (Cat No: MA5-13404, Thermo)
- Control IgG (Cat No:5415S, CST)
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