A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters |
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| Authors: | F Valtorta W Schiebler R Jahn B Ceccarelli P Greengard |
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| Affiliation: | 1. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, USA;2. Department of Medical Pharmacology, CNR Center of Cytopharmacology, University of Milano, 20129 Milano, Italy;3. Center for the Study of Peripheral Neuropathies and Neuromuscular Diseases, University of Milano, 20129 Milano, Italy;1. Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing Medical University, Nanjing, China;2. Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China;3. Southeast University, School of Public Health, Ministry of Education, Key Laboratory of Environmental Medicine and Engineering, 87 Dingjiaqiao Street, Nanjing, China;4. Department of General Surgery, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, China;5. Core Laboratory, Nantong Tumor Hospital, Nantong, China;6. Department of General Surgery, Yixing Cancer Hospital, Yixing, China;7. Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China;8. Department of General Surgery, Huai-An First People''s Hospital Affiliated to Nanjing Medical University, Huai-An, China;9. Department of Molecular Cell Biology and Toxicology, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China;1. Istituto di Endocrinologia ed Oncologia Sperimentale, CNR, Naples, Italy;1. Division of Nephrology and Hypertension, Clinical Hypertension Program, University Hospitals Case Medical Center, Cleveland, OH, USA;2. Frances Payne Bolton School of Nursing, Case Western Reserve University, Cleveland, OH, USA;3. Department of Biostatistics, Wake Forest School of Medicine, Winston-Salem, NC, USA;4. Department of Internal Medicine, Section on Nephrology, Wake Forest School of Medicine, Winston-Salem, NC, USA;5. Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA;6. Wake Forest School of Medicine, Section on Geriatrics and Gerontology, Winston-Salem, NC, USA;7. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA;8. Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX, USA;9. Nephrology Department, University of Miami Miller School of Medicine, Miami, FL, USA;10. Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA;11. Department of Internal Medicine, Wake Forest School of Medicine, Wake Forest Baptist Health, Winston-Salem, NC, USA;12. Memphis Veterans Affairs Medical Center, Memphis, TN, USA;13. Department of Internal Medicine, Northeast Ohio Neighborhood Health Services, Inc, Cleveland, OH, USA;14. Department of Medicine, Nephrology Division, Vanderbilt University, Nashville, TN, USA;15. Department of Epidemiology and Prevention, Section on Cardiovascular Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA;p. Department of Nephrology and Hypertension, Case Western Reserve University, School of Medicine, Cleveland, OH, USA;1. School of Materials Science & Engineering, Nanyang Technological University, Singapore;2. Department of Chemical Engineering, Curtin University, Sarawak, Malaysia;3. Department of Chemical Engineering, University of Tennessee, Chattanooga, TN, United States |
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| Abstract: | A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides. |
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