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 pu...
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| Veröffentlicht in: | Proteomics (Weinheim) 2005-06, Vol.5 (9), p.2417-2421 |
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| container_title | Proteomics (Weinheim) |
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| creator | Peirce, Matthew J. Begum, Shajna Saklatvala, Jeremy Cope, Andrew P. Wait, Robin |
| description | 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. |
| doi_str_mv | 10.1002/pmic.200401176 |
| format | Article |
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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.</description><identifier>ISSN: 1615-9853</identifier><identifier>EISSN: 1615-9861</identifier><identifier>DOI: 10.1002/pmic.200401176</identifier><identifier>PMID: 15887181</identifier><language>eng</language><publisher>Weinheim: WILEY-VCH Verlag</publisher><subject>Analytical, structural and metabolic biochemistry ; Animals ; Antibodies ; Biological and medical sciences ; Biotinylation ; Cell Compartmentation ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fundamental and applied biological sciences. 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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.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Biological and medical sciences</subject><subject>Biotinylation</subject><subject>Cell Compartmentation</subject><subject>Cell Line</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hybridomas - metabolism</subject><subject>Immunoprecipitation</subject><subject>Isoelectric Focusing</subject><subject>Macrophages - metabolism</subject><subject>Mass Spectrometry</subject><subject>Membrane protein</subject><subject>Membrane Proteins - isolation & purification</subject><subject>Membrane Proteins - metabolism</subject><subject>Mice</subject><subject>Miscellaneous</subject><subject>Phosphoproteins - isolation & purification</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Phosphotyrosine - immunology</subject><subject>Phosphotyrosine - metabolism</subject><subject>Protein Tyrosine Phosphatases - antagonists & inhibitors</subject><subject>Proteins</subject><subject>Proteome - metabolism</subject><subject>Solution phase isoelectric focussing</subject><subject>T-Lymphocytes - metabolism</subject><subject>Tyrosine phosphorylation</subject><subject>Vanadates - pharmacology</subject><issn>1615-9853</issn><issn>1615-9861</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkMtPGzEQh62KqkDaK0e0F7ht8Hh3_ThCRFKkAH1Q9WjZXrs17CPYu4L893WUKPTGwbI1_mbmpw-hE8BTwJhcrFpvpgTjEgMw-gEdAYUqF5zCwf5dFYfoOMZHjIFxwT6hQ6g4Z8DhCN09vPR5HNQfmynnfOeHdbYag3feqMH3Xeb6kPmuHo3XTfr628d0wrpRg62z1rY6qM5mq9AP1nfxM_roVBPtl909Qb_m1w-zr_nyfnEzu1zmpiwJzRnjjinQNaWKEc1Lx51z2mjlLOHOlqQQoi6pEsQxo1NZA1hqKloxZRgpJuh8Ozctfh5tHGTro7FNk8L0Y5SUCSAVlO-CIERJCaUJnG5BE_oYg3VyFXyrwloClhvVcqNa7lWnhtPd5FG3tn7Dd24TcLYDVDSqcUmU8fGNo1zQQmwiii334hu7fmet_HZ7M_s_RL7t9XGwr_teFZ6SgoJV8vfdQrKrxc_vc7KQP4p_pTSpWQ</recordid><startdate>20050601</startdate><enddate>20050601</enddate><creator>Peirce, Matthew J.</creator><creator>Begum, Shajna</creator><creator>Saklatvala, Jeremy</creator><creator>Cope, Andrew P.</creator><creator>Wait, Robin</creator><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><general>Wiley-VCH</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20050601</creationdate><title>Two-stage affinity purification for inducibly phosphorylated membrane proteins</title><author>Peirce, Matthew J. ; Begum, Shajna ; Saklatvala, Jeremy ; Cope, Andrew P. ; Wait, Robin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4426-778f7a1bd66a72b84f8fffbcbafe28fe42399d46a92f7cbbafb11e6c5657ac723</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Biological and medical sciences</topic><topic>Biotinylation</topic><topic>Cell Compartmentation</topic><topic>Cell Line</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fundamental and applied biological sciences. 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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Analytical, structural and metabolic biochemistry Animals Antibodies Biological and medical sciences Biotinylation Cell Compartmentation Cell Line Electrophoresis, Polyacrylamide Gel Fundamental and applied biological sciences. Psychology Hybridomas - metabolism Immunoprecipitation Isoelectric Focusing Macrophages - metabolism Mass Spectrometry Membrane protein Membrane Proteins - isolation & purification Membrane Proteins - metabolism Mice Miscellaneous Phosphoproteins - isolation & purification Phosphoproteins - metabolism Phosphorylation Phosphotyrosine - immunology Phosphotyrosine - metabolism Protein Tyrosine Phosphatases - antagonists & inhibitors Proteins Proteome - metabolism Solution phase isoelectric focussing T-Lymphocytes - metabolism Tyrosine phosphorylation Vanadates - pharmacology |
| title | Two-stage affinity purification for inducibly phosphorylated membrane proteins |
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