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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >Large-Surface Biosensor Technology for Enhanced Recovery in Protein Characterization
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Large-Surface Biosensor Technology for Enhanced Recovery in Protein Characterization

机译：大表面生物传感器技术可提高蛋白质表征的回收率

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摘要

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm2). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-μl segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.
机译：通过回收针对人胰岛素原C肽的单克隆抗体，测试了使用表面等离振子共振（SPR）的大表面生物传感器技术的蛋白纯化。值得注意的是，显示了从多组分蛋白质混合物可逆地附着/解吸和实际纯化抗体。对于肽配体的初始芯片连接，将C-肽进行生物素化，然后将其附着到金表面较大（有效面积26 mm 2 ）的塑料芯片上的中性亲和素上。实时SPR监测抗体的结合和解吸，并采用不同的洗脱条件。气泡之间60μl区域中的5％甲酸（与芯片表面接触3分钟）对于随后的分析非常有效。以这种方式，在单个捕获/洗脱循环中回收的蛋白质量高达35 pmol。通过SDS-PAGE评估表明，在此洗脱过程中，各馏分之间基本没有残留，纯化后混合物中的其他蛋白质也没有残留。与现有的商用仪器相比，该技术具有更高的回收率，并可以监测监控蛋白的结合/解吸。已证明可以在SPR方法中纯化蛋白质，从而在多摩尔水平上回收亲和性伴侣。

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