Construction of the FLAG-RIOK3 expression construct is detailed in the supplementary methods . Immunoprecipitation was done using anti-FLAG® M2 magnetic beads (Sigma-Aldrich). Antigen and bound interacting species were eluted using either FLAG peptide or heating the beads to 70°C for 10 min in denaturing LDS buffer. Gels were stained using ProteoSilver™ Plus Silver Stain Kit (Sigma-Aldrich). Bands were excised from the gel, digested with trypsin and subjected to tandem mass spectrometry (LC-MS/MS) analysis as described previously.40 (link)
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Immunoprecipitation and Mass Spectrometry
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Other organizations : John Radcliffe Hospital, University of Oxford, Karolinska Institutet, Linköping University, University of Leicester
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Variable analysis
independent variables
- Construction of the FLAG-RIOK3 expression construct
- Antigen and bound interacting species
- Anti-FLAG® M2 magnetic beads (Sigma-Aldrich)
- FLAG peptide
- Heating the beads to 70°C for 10 min in denaturing LDS buffer
- ProteoSilver™ Plus Silver Stain Kit (Sigma-Aldrich)
- Not explicitly mentioned
- Not explicitly mentioned
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