BIACORE 2000

一组7个合格的主要用户详细介绍了广泛的实验计划，具体采用BIAcore分析，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噬菌体显示肽与传感器芯片C1和/或F1的结合来确定，这些芯片与重组IgE受体外结构域或IgE共价衍生；（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传感器芯片来模拟细胞-细胞TCR- mhc /肽介导的相互作用，以确定脂质单层中TCR-配体的识别；（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NADPH氧化酶，它由一个完整的膜蛋白、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质和膜因子的分子相互作用（单独和组合），确定细胞质和膜因子的分子相互作用（单独和组合），确定细胞质和膜因子的分子相互作用（单独和组合），以及确定细胞质和膜因子的分子相互作用（单独和组合），以及确定细胞质和膜因子的分子相互作用（单独和组合），并确定它们的亲和力如何受到阴离子脂质、离子强度和GTP的影响；（iv） Rac在上述系统中的确切作用尚不清楚，但目前的一种假设是gtp结合的Rac与p67phox的关联增加了该蛋白对黄细胞色素的亲和力。这将通过测定p67phox在分析溶液中存在和不存在Rac-GTP的情况下与固定化、纯化、再固化的黄细胞色素结合的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锌指蛋白TFIIA及其截断或突变的衍生物，在固定在BIAcore芯片上的5S DNA或5S RNA溶液中确定；（六）利用表面等离子体共振技术研究成纤维细胞生长因子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细胞质结构域的亲和力。现在将使用BIAcore系统分析2种混合分析中的结合组分，以确定动力学结合参数。（vii）自身抗体结合野生型和突变型纤维蛋白，在存在和不存在汞，使用BIAcore机器。然后将评估固定在生物传感器芯片上的纤维蛋白的抗体k/on和解离（k/off）的hg依赖性变化的定量。

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.