Source and avoidance of the "nothing dehydrogenase" effect, a spurious band produced during polyacrylamide gel electrophoresis of dehydrogenase enzymes from yeasts.
During a study of the distribution of several NAD-linked dehydrogenase enzymes in various yeasts, in which polyacrylamide gel electrophoresis, followed by activity staining with phenazine methosulfate and a tetrazolium was used, a band was frequently detected, the production of which appeared to be independent of any added substrate (the "nothing dehydrogenase" effect). It has been shown that this effect is caused by alcohol dehydrogenase acting on traces of ethanol inadvertently introduced into the system. Two sources of ethanol were identified. They were (i) the enzyme extracts, which could be freed from ethanol by gel filtration, and (ii) the acrylamide used to prepare the gel, which could be freed from ethanol by recrystallization from ethanol-free chloroform. It is suggested that the use of commercial chloroform (stabilized with ethanol) as a recrystallizing solvent is the source of ethanol contamination in commercial preparations of acrylamide.
D020106:Acrylamide; D000178:Acrylamides / Q000302:isolation & purification; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000302:isolation & purification; D010088:Oxidoreductases / Q000032:analysis*; D012997:Solvents; D013194:Staining and Labeling; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology*
D000178:Acrylamides; D012997:Solvents; D020106:Acrylamide; D000431:Ethanol; D010088:Oxidoreductases