Generic method for quantification of FLAG-tagged fusion proteins by a real time biosensor |
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| Institution: | 3. Departments of Molecular Physiology and Biophysics and University of Vermont, Burlington, Vermont 05405;4. Departments of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405;3. From the Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232 and;4. Division of Medicinal Chemistry, The University of Texas at Austin, Austin, Texas 78712;1. Water Research and Development Centre (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain;2. Instituto de Estudios Biofuncionales, Departamento de Química-Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain;3. Instituto de Recursos Naturales y Agrobiologia (IRNASA-CSIC), 37008 Salamanca, Spain;4. Departamento de Bioquimica y Biologia Molecular, Universidad Complutense de Madrid, 28040 Madrid, Spain;5. Department of Biochemistry, Microbiology and Biotechnology, Far Eastern Federal University, 690600 Vladivostok, Russia;6. Departamento de Bioquimica y Biologia Molecular, Universidad de Salamanca, 37007 Salamanca, Spain;7. Departamento de Quimica Física, Universidad de Salamanca, 37008 Salamanca, Spain;8. Chembiotech Laboratorios, Kyrewood House, Tenbury Wells, Worcestershire WR15 8SG, UK;1. University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Biology, A?ker?eva 7, SI-1000 Ljubljana, Slovenia;2. Jo?ef Stefan Institute, Department of Biotechnology, Jamova 39, SI-1000 Ljubljana, Slovenia |
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| Abstract: | Availability of rapid quantitative protein-expression analysis is often the bottleneck in high throughput screening applications. A real time biosensor was employed to establish a quantitative assay for FLAG fusion proteins using FLAG-tagged bacterial alkaline phosphatase as standard. A range of FLAG-tagged bacterial alkaline phosphatase concentrations were injected over the anti-FLAG M2 antibody surface of the biosensor and used as standards to determine the concentration of different FLAG-tagged proteins with a molecular mass of 18.1 kDa respectively 49.3 kDa from yeast culture supernatants. The M2 immobilized chip was found to retain binding capacity following regeneration for at least 120 cycles. This real time biosensor method allows the quantitation of proteins from culture supernatants using a calibration curve obtained with a different protein. Further benefits include the short assay time of approximately 5 min, the small amount of sample required (35 μl per injection) and the ability to monitor the binding event in real time. |
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