title: Two-stage affinity purification for inducibly phosphorylated membrane proteins.
abstract: Characterisation of tyrosine phosphorylations induced in immune cells in response to inflammatory stimuli may help elucidate the molecular bases of the diversity of immune responses. We have used anti-phosphotyrosine antibodies in combination with cell surface biotinylation in a two-step affinity purification procedure to recover pervanadate-induced tyrosine phosphorylated proteins from sub-cellular compartments, including the cell surface, of murine T cells and macrophages prior to separation by solution-phase isoelectric focussing and one-dimensional gel electrophoresis and identification by tandem mass spectrometry.
authors.affiliation: Kennedy Institute of Rheumatology Division, Imperial College, London, UK. m.peirce@imperial.ac.uk
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Peirce
authors.forename: Shajna
authors.initials: S
authors.lastname: Begum
authors.forename: Jeremy
authors.initials: J
authors.lastname: Saklatvala
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Cope
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
mesh_terms: D000818:Animals; D000906:Antibodies; D019921:Biotinylation; D002451:Cell Compartmentation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006825:Hybridomas / Q000378:metabolism; D047468:Immunoprecipitation; D007525:Isoelectric Focusing; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D020543:Proteome / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D000906:Antibodies; D008565:Membrane Proteins; D010750:Phosphoproteins; D020543:Proteome; C076715:pervanadate; D019000:Phosphotyrosine; D014638:Vanadates; D017027:Protein Tyrosine Phosphatases