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Baldin_Guide_answers - Guide for Baldin et al(1993 Cyclin...

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Guide for Baldin et al. (1993) Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes & Dev. 7:812-821. These questions are intended to help you read the paper in an inquisitive fashion and to help you focus on some of the issues you should understand. Please cover each of these questions during your reading and studying. Please submit the answers to 5 of these questions for your homework assignment. 1) To investigate the role of cyclin D1 in vivo, the authors obtain a cyclin D1 cDNA clone. They first use this clone to overexpress and purify cyclin D1 from bacterial cells (this is called recombinant cyclin D1) and produce an anti-recombinant cyclin D1 antibody. Second, they immunoprecipitate (IP) protein from human cells using this antibody. Figure 1 presents their data demonstrating that the protein they IP from human cells is indeed cyclin D1. How does the experiment in Figure 1A, lane 6 (there is no protein in the IP in lane 6) demonstrate the specificity of their antibody for cyclin D1? The authors IP a protein from cells that they believe is cylcin D1. To show that it is indeed cyclin D1, they preadsorb their antibody serum with immobilized recombinant cylcin D1. The resulting serum should no longer have any cyclin D1-specific antibodies. Correspondingly, the IP with this preadsorbed antibody no longer precipitates the cellular D1, demonstrating that their antibody is not immunoprecipitating some superfluous protein by chance. 2) The authors have also produced cyclin D1 protein by transcribing and translating their cDNA clone in vitro. (RNA was produced from the plasmid DNA probably by using one of the commercially available phage polymerases such as SP6 or T7. The RNA was then translated in a rabbit reticulocyte lysate in the presence of 35S-methionine.) How do they use this labeled in vitro translated protein to demonstrate that the immunoprecipitated protein is indeed cyclin D1 (see Figure 1C)? I.e., what is the comparison they are making in Fig 1C and how do they carry it out?
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