Biochemistry, 4th Edition P42. Continuing Garrett and Grisham's innovative conceptual and organizing framework, "Essential Questions," BIOCHEMISTRY guides students through course concepts in a way that reveals the beauty and usefulness of biochemistry in the everyday world. Streamlined for increased clarity and readability, this edition also includes new photos and illustrations that show the subject matter consistently throughout the text. New end-of-chapter problems, MCAT practice questions, and the unparalleled text/media integration with the power of CengageNOW round out this exceptional package, giving you the tools you need to both master course concepts and develop critical problem-solving skills you can draw upon. | What Is the Polymerase Chain Reaction PCR 373 that transcription of a reporter gene driven by the GAL4 promoter can take place Figure . Protein X fused to the GAL4-DNA-binding domain DB serves as the bait to fish for the protein Y target and its fused GAL4 TA domain. This method can be used to screen cells for protein targets that interact specifically with a particular bait protein. To do so cDNAs encoding proteins from the cells of interest are inserted into the TA-containing plasmid to create fusions of the cDNA coding sequences with the GAL4 TA domain coding sequences so a fusion protein library is expressed. Identification of a target of the bait protein by this method also yields directly a cDNA version of the gene encoding the target protein. Identifying Protein-Protein Interactions Through Immunoprecipitation If antibodies against one protein of a multiprotein complex are available the entire complex can be immunoprecipitated and its composition analyzed. Attachment of such antibodies to glass or agarose beads which easily sediment in a centrifuge makes recovery of the complex very simple. Because antibodies against it are commercially available the hemagglutinin HA peptide sequence YPYDVPDYA is a useful protein fusion tag not only for fusion protein purification Table but also for analysis of protein-protein interactions. Expressing an HA-tagged protein in vivo followed by immunoprecipitation allows the isolation of protein complexes of which the HA-tagged protein is a member. The other members of the complex can then be identified to establish the various interacting partners within the multiprotein complex. What Is the Polymerase Chain Reaction PCR Polymerase chain reaction or PCR is a technique for dramatically amplifying the amount of a specific DNA segment. A preparation of denatured DNA containing the segment of interest serves as template for DNA polymerase and two specific oligonucleotides serve as primers for DNA synthesis as
