Two-stage affinity purification for inducibly phosphorylated membrane proteins

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.