107 416B cells were lysed in the whole cell lysis buffer (50 mM Tris-HCl pH=8, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA), supplemented with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). Cell were homogenized and lysate cleared by centrifugation. UTX immunoprecipitation was performed in the whole cell lysis buffer with 16 ug of antibody bound to 100ul of Dynabeads Protein G (Thermo Fisher Scientific) and incubated for 1,5h at 4°C with rotation. IP was washed five times with IP wash buffer (10 mM Tris-HCl pH=8, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA), supplemented with protease inhibitor (Sigma). UTX immunoprecipitates were eluted by boiling in 1x LDS loading buffer, reduced with 5 mM TCEP, alkylated with 10 mM iodoacetamide and electrophoresed in Novex NuPAGE Bis-Tris 4%–12% gels (Life Technologies). Gels were stained with colloidal Coomassie (Sigma). Whole lanes were cut in slices and samples processed for MS analysis as described previously65 (link).
Protocol detail
Proteomic Analysis of UTX Interactome
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Antibody
Bis tris
Buffer
Cell
Centrifugation
Edta
Gels
Immunoprecipitation
Inhibitors
Iodoacetamide
Nacl
Np 40
Phosphatase
Protease inhibitor
Protein g
Tcep
Tris
Corresponding Organization :
Other organizations : Wellcome Sanger Institute, Wellcome/MRC Cambridge Stem Cell Institute, University of Cambridge, Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria, Medical Research Council, Universität Ulm, German Cancer Research Center, Heidelberg University, Cambridge University Hospitals NHS Foundation Trust
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Variable analysis
independent variables
- Concentration of UTX antibody (16 ug)
- Proteins co-immunoprecipitated with UTX
- Whole cell lysis buffer (50 mM Tris-HCl pH=8, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA)
- Supplementation with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors (Sigma)
- IP wash buffer (10 mM Tris-HCl pH=8, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA) with protease inhibitor (Sigma)
- Elution by boiling in 1x LDS loading buffer, reduction with 5 mM TCEP, and alkylation with 10 mM iodoacetamide
- Electrophoresis in Novex NuPAGE Bis-Tris 4%–12% gels (Life Technologies)
- Colloidal Coomassie staining of gels
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