Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

Quantitative Application for SDS–PAGE in a Biochemistry Lab

“…Polyacrylamide gel electrophoresis (PAGE) is commonly conducted in the presence of sodium dodecyl sulfate (SDS–PAGE) to resolve polypeptides for two types of analysis: (i) to determine the molecular mass of proteins and (ii) to analyze the subunit composition of a given protein. , An often overlooked capability of SDS–PAGE is that it can be used to quantitate the concentration of a single protein from a heterogeneous mixture of proteins, which is commonly accomplished by ELISA (enzyme-linked immunosorbant assay) or Western blotting. The experiment described, which is amenable to modification for a wide range of applications, combines the generation of a standard curve of bovine serum albumin (BSA), as described by Barufaldi et al, with SDS–PAGE to determine the concentration of a single protein, serum albumin, from a sample of whole human serum.…”

Program for Simulating Gel Electrophoresis of Enzyme-Digested Proteins

“…1 Nash showed students how to use SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase. 2 Saperas and Fonfri ́a-Subiros tested students' abilities to find out which granules in a detergent contained proteolytic enzymes for cleaning. 3 Usher and Barrette-Ng developed a Java applet for students to simulate the separation of proteins by ion-exchange chromatography and the SDS-PAGE of one of its bands to determine whether it contained one or more proteins.…”

“…For example, Evans and Torre used one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to demonstrate how to separate protein mixtures in several snake venoms . Nash showed students how to use SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase . Saperas and Fonfría-Subiros tested students’ abilities to find out which granules in a detergent contained proteolytic enzymes for cleaning .…”

The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

“…Alcohol dehydrogenase (ADH) is an ideal enzyme for the undergraduate biochemistry laboratory. − It is purified relatively easily from yeast − and liver, is commercially available in purified form, and has been extensively studied and reviewed. − The enzyme has two substrates, alcohol and NAD + , and is therefore more interesting to work with than the single-substrate or pseudo-first order enzymes that are typically studied in undergraduate laboratories . An added advantage is that NADH, the reduced product, is a UV chromophore that is easily assayed spectrophotometrically.…”