SGER: Development and Testing of a "Generic" K+ Channel Antibody

[320336] Berkowitz, Gerald a .对K+通道分子特性的研究受到了阻碍，因为缺乏一种探针可以选择性地识别膜制剂和cDNA翻译产物中的这些蛋白质。一项研究计划被启动，旨在通过产生抗体来开发这种探针，该抗体对应于蛋白质孔中推断的氨基酸序列，该序列作为选择性过滤器，在已知的K+通道中高度保守。这项工作的目的是通过检查抗体对广泛的K+通道的亲和力来评估抗体的有效性，以及它对这类蛋白质的选择性。这项工作将包括免疫印迹分析抗体与天然膜（叶绿体内膜）中已知的K+通道的反应性，以及在表达K+通道cDNA产品的异源系统中（酵母中的KAT1 K+通道cDNA，肾细胞系中的drkl cDNA，以及大肠杆菌中的Shab， Shaw， Shal和KAT1 cDNA）。该实验室使用了一种不同寻常的技术来培养针对所有已知K+通道共同的K+通道蛋白的特定部分的特异性抗体。该项目的目标是证明这种抗体是“通用的”，这意味着它可以识别所有的K+通道，而不管它用于什么系统。由于目前普遍认为抗体生产中使用的方法不起作用，因此无法产生特异性探针，因此需要这样的证明。这是一项“高风险、高回报”的开创性研究，非常适合探索性R研究项目的小额赠款。* * *​}!!!F (Times New Roman Symbol & Arial " h " $" 5 Geza Hrazdina博士。Geza Hrazdina博士

9320336 Berkowitz, Gerald A. Progress toward the molecular characterization of K+-channels is hindered by lack of a probe which can selectively identify these proteins in membrane preparations, and in translation products of cDNA's. A research program was initiated and aimed at developing such a probe by generating antibodies to a short peptide corresponding to the deduced amino acid sequence in the pore of the protein which acts as a selectivity filter, and is highly conserved in known K+ channels. The objective of the work is to evaluate the usefulness of the antibody by examining it's affinity for a broad range of K+ channels, and it's selectivity for this class of proteins. The work will involve immunoblot analysis of the antibody's reactivity with known K+ channels in native membranes (chloroplast inner envelope) and in heterologous systems expressing products of K+ channel cDNA's (KAT1 K+ channel cDNA in yeast, drkl cDNA in kidney cell lines, and Shab, Shaw, Shal and KAT1 cDNAs in E. coli). %%% The laboratory made use of an unusual technique to raise a specific antibody to a specific part of the K+ channel protein that is common to all known K+ channels. The goal of the project is to show proof that this antibody is "generic", that means that it recognizes all K+ channels, irrespective of the system it is used in. Since it is presently generally assumed that the method used in the antibody production will not work, and therefore no specific probes can be generated, such a proof is necessary. This is a "high risk, high reward" pioneering study that is well suited to the Small Grants for Exploratory R esearch program. *** } ! ! ! F ( Times New Roman Symbol & Arial " h " $" 5 Dr. Geza Hrazdina Dr. Geza Hrazdina