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  Home > JCE Print > Journal of Chemical Education > Issues > 2002  > November  >
In the Laboratory
Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates
Karlett Parra-Belky
Department of Chemistry, Ball State University, Muncie, IN 47306
Cover
November 2002
Vol. 79 No. 11
p. 1348
Abstract
A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.
Supplement
Instructions for the students and notes for the instructor are available.
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*  Citation
 Parra-Belky, Karlett. J. Chem. Educ. 2002 79 1348.
*  Keywords
 Biochemistry; Biotechnology; Electrophoresis; Enzymes; Laboratory Instruction; Proteins / Peptides; Separation Science
*  History
 Created:
Last Updated:		 September 30, 2002
March 16, 2005
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