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Two‐stage affinity purification for inducibly phosphorylated membrane proteins
Author(s) -
Peirce Matthew J.,
Begum Shajna,
Saklatvala Jeremy,
Cope Andrew P.,
Wait Robin
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401176
Subject(s) - biotinylation , phosphorylation , isoelectric focusing , tyrosine phosphorylation , tyrosine , tandem mass spectrometry , chemistry , protein phosphorylation , isoelectric point , biochemistry , tandem affinity purification , affinity chromatography , cell , membrane protein , immune system , biology , mass spectrometry , membrane , chromatography , protein kinase a , immunology , enzyme
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.