BIACORE 2000

比亚科2000

• 批准号: 2766839
• 负责人: MICHAEL W ROBERTSON
• 金额: $ 25.82万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2766839
• 关键词: biomedical equipment; biomedical equipment purchase; surface plasmon resonance

A group of seven qualifying Primary Users have detailed a broad range of experimental plans that specifically employ BIAcore analysis as summarized herein: (i) Characterization of association and dissociation rate constants for the interaction of immunoglobulin E (IgE) and the high affinity IgE receptor (FcepsilonRI) is a major goal of the PI. The effect of point mutations on alterations of k/on and/or k/off values will be determined by measurement of phage-displayed peptide binding to sensor chips C1 and/or F1 covalently derivatized with either recombinant IgE receptor ectodomain or IgE; (ii) Determination of the kinetics of the interaction between the T cell receptor (TCR) and its ligands, specifically MHC class I-peptide complexes. To extend our recently published study of peptide/Class I-TCR binding kinetics, which showed a correlation between the affinity of TCR for particular ligands and the biological outcome, we plan to determine TCR-ligand recognition in lipid monolayers using the HPA sensor chip to model cell-cell TCR-MHC/peptide mediated interactions; (iii) The superoxide generating system of neutrophils is a membrane-associated enzyme complex, NADPH oxidase, which consists of an integral membrane protein, flavocytochrome b245 and cytosolic proteins p47phox, p67phox and Rac. BIAcore analysis will be used to study the molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how their affinity is affected by anionic lipids, ionic strength and GTP; (iv) The precise role of Rac in the foregoing system is still unknown, but one current hypothesis is that the association of GTP-bound Rac with p67phox increases the affinity of this protein for the flavocytochrome. This will be tested by determining the kinetics and affinity of binding of p67phox to immobilized, purified, relipidated flavocytochrome in the presence and absence of Rac-GTP in the analyze solution; (v) The energetic processes that drive the binding reactions for DNA and RNA will be determined using the zinc finger protein TFIIA, and truncated or mutated derivatives, in solution with either 5S DNA or 5S RNA immobilized on a BIAcore chip; (vi) Surface plasmon resonance technology will be utilized to study fibroblast growth factor (FGF) receptor- mediated signal transduction. Several proteins, identified using the two-hybrid system, show affinity for the cytoplasmic domain of FGFR-1. The binding components from the 2 hybrid analysis will now be analyzed using the BIAcore system to determine the kinetic binding parameters. (vii) Autoantibody binding to wildtype and mutant fibrillarin, in the presence and absence of mercury, using the BIAcore machine. Quantitation of Hg-dependent changes of antibody k/on and dissociation (k/off) from fibrillarin immobilized on the biosensor chip will then be assessed.

由七名合格的主要用户组成的小组详细介绍了专门采用 BIAcore 分析的广泛实验计划，如下所述： (i) 免疫球蛋白 E (IgE) 和高亲和力 IgE 受体 (FcepsilonRI) 相互作用的结合和解离速率常数的表征是 PI 的主要目标。点突变对 k/on 和/或 k/off 值改变的影响将通过测量与重组 IgE 受体胞外域或 IgE 共价衍生的传感器芯片 C1 和/或 F1 结合的噬菌体展示肽来确定； (ii) 确定 T 细胞受体 (TCR) 与其配体（特别是 MHC I 类肽复合物）之间相互作用的动力学。为了扩展我们最近发表的肽/I 类-TCR 结合动力学研究，该研究显示了 TCR 对特定配体的亲和力与生物学结果之间的相关性，我们计划使用 HPA 传感器芯片来确定脂质单层中的 TCR-配体识别，以模拟细胞间 TCR-MHC/肽介导的相互作用； (iii) 中性粒细胞的超氧化物生成系统是一种膜相关酶复合物，即 NADPH 氧化酶，它由整合膜蛋白、黄细胞色素 b245 和胞质蛋白 p47phox、p67phox 和 Rac 组成。 BIAcore 分析将用于研究细胞溶质和膜因子单独和组合的分子相互作用，并确定细胞溶质和膜因子单独和组合的分子相互作用如何，确定细胞溶质和膜因子单独和组合的分子相互作用如何，并确定细胞溶质和膜因子单独和组合的分子相互作用如何， 并确定阴离子脂质、离子强度和 GTP 如何影响它们的亲和力； (iv) Rac 在上述系统中的精确作用仍然未知，但目前的一个假设是，GTP 结合的 Rac 与 p67phox 的结合增加了该蛋白质对黄素细胞色素的亲和力。这将通过在分析溶液中存在和不存在 Rac-GTP 的情况下测定 p67phox 与固定、纯化、再脂化的黄素细胞色素的结合动力学和亲和力来进行测试； (v) 驱动 DNA 和 RNA 结合反应的能量过程将使用固定在 BIAcore 芯片上的 5S DNA 或 5S RNA 溶液中的锌指蛋白 TFIIA 和截短或突变的衍生物来确定； (vi) 表面等离子共振技术将用于研究成纤维细胞生长因子（FGF）受体介导的信号转导。使用双杂交系统鉴定的几种蛋白质显示出对 FGFR-1 胞质结构域的亲和力。现在将使用 BIAcore 系统分析来自 2 次混合分析的结合成分，以确定动力学结合参数。 (vii) 使用 BIAcore 机器，在存在和不存在汞的情况下，自身抗体与野生型和突变型纤维蛋白结合。然后将评估固定在生物传感器芯片上的原纤维蛋白的抗体 k/on 和解离 (k/off) 的 Hg 依赖性变化的定量。