Generic method for quantification of FLAG-tagged fusion proteins by a real time biosensor
Generic method for quantification of FLAG-tagged fusion proteins by a real time biosensor
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DOI：
10.1016/j.jbbm.2007.01.004
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发表时间：
2007-06-10
影响因子：
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通讯作者：
Jungbauer, Alois
中科院分区：
文献类型：
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作者：
Mersich, Christa;Jungbauer, Alois
快速定量蛋白表达分析的可用性往往是高通量筛选应用的瓶颈。采用实时生物传感器，以FLAG标记的细菌碱性磷酸酶为标准，建立了FLAG融合蛋白的定量检测方法。在生物传感器的抗flag M2抗体表面注射一系列flag标记的细菌碱性磷酸酶浓度作为标准，测定酵母培养上清中不同flag标记蛋白的浓度，这些蛋白的分子量分别为18.1 kDa和49.3 kDa。发现M2固定芯片在再生至少120次循环后仍保持结合能力。这种实时生物传感器方法允许使用不同蛋白质获得的校准曲线从培养上清中定量蛋白质。进一步的好处包括检测时间短，约为5分钟，所需样品量少（每次注射35 μ l），并且能够实时监测结合事件。(C) 2007 Elsevier B.V.版权所有
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 mu l per injection) and the ability to monitor the binding event in real time. (C) 2007 Elsevier B.V. All rights reserved.