To monitor phosphorylation and ubiquitination of RIPK1, we used TNF-Flag treatment.28 (link) Liver tissue was homogenised in buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Potassium hydroxide pH7.5, 0.2% NP-40, 120 mM NaCl, 0.27M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM Na3VO4, cOmpleteTM Protease Inhibitor Cocktail, 2 mM phenylmethylsulfonyl fluoride and 10 mM N-Ethylmaleimide. Samples were either incubated with anti-FLAG M2 magnetics beads (Sigma) for 4 hours or RIPK1 antibody (Cell Signaling) overnight followed by 2 hours incubation with magnetics beads (Bio-rad) at 4°C. The beads were washed and proteins were eluted in 70°C with sample buffer and the eluted proteins were fractionated by SDS-PAGE gels. Proteins were detected by immunoblotting as described above.
Anti flag m2 magnetics beads
Manufactured by Merck Group
Anti-FLAG M2 magnetic beads are a laboratory tool used for the purification and detection of proteins tagged with the FLAG epitope. They consist of magnetic beads coated with the Anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag sequence. These beads provide a simple and efficient method for isolating and concentrating FLAG-tagged proteins from complex samples.
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2 protocols using anti flag m2 magnetics beads
1
Immunoprecipitation of RIPK1 Phosphorylation
2
Investigating CSB-PGBD3 Interactions under Stress
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HEK293FT cells were transiently transfected with wild type and the 3 mutant p3XFLAG-CSB-PGBD3 plasmids using Lipo2000 (Invitrogen) and cultured for 24 h. Cells were then divided into three groups: without treatment; treatment with ultraviolet light (UV) irradiation (10 J/m2); treatment with H2O2 (5mM, 4°C, 10min). After treatment, protein of the cells was extracted using NETN buffer, and pulled down with anti-FLAG M2 magnetics beads (Sigma-Aldrich M8823). The interaction between CSB-PGBD3 and RNApol II was analyzed by western blot with the antibody against FLAG (Cell Signaling Technology 2368) and RNApol II (Santa Cruz Biotechnology sc-9001). All steps were performed at 4°C.
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