| Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR
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Author:
Date:
2014-11-05
[Abstract] Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the ...
[摘要] 表面等离子体共振（SPR）被广泛用于产生关于生物分子之间的特异性相互作用的动力学和亲和力信息。这种技术是无标签的，并实时监控绑定事件。它通常用于表征单克隆抗体 - 抗原相互作用。该方案特别描述了使用SPR与Biacore T100仪器测量粗杂交瘤样品对蛋白质的亲和力。为此，首先通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上（Canziani等人，2004; Schraml和Biehl，2012）。然后来自杂交瘤上清液的抗体通过其Fc区非共价地捕获到表面上，提供最佳的分析物结合方向。最后，通过与EDC/NHS交联来稳定所得的复合物，以避免在测量和再生期间的基线漂移（Pope等人，2009）。然后在若干浓度下监测与蛋白质的相互作用，并且用从经典动力学分析获得的相应K D确定其对固定化抗体的亲和力。这种设置避免了二价抗体的亲合力效应，允许使用具有未知浓度的非纯化分析物和以类似的稳定的共价定向方式特异性捕获抗体。
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| Protein-lipid Interaction Analysis by Surface Plasmon Resonance (SPR)
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Author:
Date:
2014-09-20
[Abstract] Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and rapid determination of association and dissociation rates using small amounts of samples. An extensive range of binding analyses can be performed by SPR such as protein–protein, protein–membrane (lipids), protein–carbohydrate, protein–nucleic acid and even protein-small molecules. This protocol describes the binding of an antimicrobial protein (used as ligand) to a lipopolysaccharide (LPS) (used as analyte) after immobilization onto a CM sensor ...
[摘要] 脂质与蛋白质的相互作用是生物膜框架中的重要事件。 蛋白质 - 脂质结合的亲和力和特异性的评估可以提供有用的信息来阐明细胞膜功能。 表面等离子体共振（SPR）是研究大分子相互作用的强大技术，允许使用少量样品直接和快速测定缔合和解离速率。 可以通过SPR进行广泛范围的结合分析，例如蛋白质 - 蛋白质，蛋白质 - 膜（脂质），蛋白质 - 碳水化合物，蛋白质 - 核酸甚至蛋白质 - 小分子。 该方案描述了在通过胺偶联固定在CM传感器芯片上之后，抗微生物蛋白（用作配体）与脂多糖（LPS）（用作分析物）的结合。
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| Protein-peptide Interaction by Surface Plasmon Resonance
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Author:
Date:
2013-02-05
[Abstract] This protocol measures the protein-peptide interaction by surface plasmon resonance (SPR) using Biacore X100 (GE Healthcare). The Biacore system can monitor the direct interaction between biomolecules. There are several methods of immobilizing a ligand to the sensor chip. The optimal immobilization method for each experiment needs to be selected. In this protocol, we employed amine coupling to immobilize the protein to the carboxyl-type sensor chip. The procedure generally follows the “Instrument Handbook” of Biacore X100.
[摘要] 该协议使用Biacore X100（GE Healthcare）通过表面等离子体共振（SPR）测量蛋白质 - 肽相互作用。 Biacore系统可以监测生物分子之间的直接相互作用。 存在将配体固定到传感器芯片的几种方法。 需要选择每个实验的最佳固定方法。 在该协议中，我们使用胺偶联固定蛋白质到羧基型传感器芯片。 该程序通常遵循Biacore X100的"仪器手册"。
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