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ERIC Number: EJ895551
Record Type: Journal
Publication Date: 2010
Pages: 8
Abstractor: As Provided
ISSN: ISSN-1470-8175
New Ideas for an Old Enzyme: A Short, Question-Based Laboratory Project for the Purification and Identification of an Unknown LDH Isozyme
Coleman, Aaron B.
Biochemistry and Molecular Biology Education, v38 n4 p253-260 Jul-Aug 2010
Enzyme purification projects are an excellent way to introduce many aspects of protein biochemistry, but can be difficult to carry out under the constraints of a typical undergraduate laboratory course. We have designed a short laboratory project for the purification and identification of an "unknown" lactate dehydrogenase (LDH) isozyme that can fit into a multiproject course without consuming too many laboratory days. The streamlined purification utilizes ammonium sulfate precipitation, affinity chromatography, and size exclusion chromatography to give good recovery of LDH with minimal equipment requirements, and can be completed in three laboratory periods of 3-4 hours. As part of this, we have designed a novel, qualitative format for an LDH activity assay that allows students to rapidly screen their column chromatography fractions without the need of a spectrophotometer or plate reader. The analysis phase of the project is question-driven, and can be completed in two laboratory periods. The students must determine which purification technique was most effective by quantifying LDH activity and total protein content at each step of the purification, and then identify their unknown isozyme through agarose gel electrophoresis. This module provides an engaging format for teaching protein biochemistry, with the flexibility to allow an instructor to modify it for their particular curriculum. (Contains 3 figures and 3 tables.)
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Publication Type: Journal Articles; Reports - Descriptive
Education Level: Higher Education
Audience: Teachers
Language: English
Sponsor: N/A
Authoring Institution: N/A