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

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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