Detection of the Coprinus cinereus protein Rad51 in meiotic and mitotic tissue and characterization of the Rad51 antibody

Abstract

Understanding the mechanisms behind DNA repair and meiotic recombination in Coprinus cinereus is an integral step in furthering the understanding of the eukaryotic cell. The proteins Rad51 and Rad50 have been shown to be required for these processes, so the primary objective of this study is to learn more about Rad51 in the hopes of using it as a tool to better understand Rad50. The Rad51 antibody was purified by means of affinity columns and a surf blot and immunoblotting was used to identify and collect the purified antibody. Once it was proven that the antibody recognized bacterially expressed Rad51, a series of protein dilutions were set up to test its sensitivity. The antibody was then used to probe a blot of C. cinereus meiotic and mitotic proteins in order to investigate the presence of Rad51 in the protein extract taken from tissue cells. After the antibody has been proven to identify Rad51 in vitro it will become a tool to mark the presence of DNA double-strand breaks and be used to determine if Rad50 is needed for DNA double-strand break initiation.