Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes.
A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.
Sock
J
J
Research Station, Agriculture Canada, Winnipeg, Manitoba.
Rohringer
R
R
D000818:Animals; D002384:Catalysis; D002417:Cattle; D003124:Colorimetry; D004798:Enzymes / Q000032:analysis*; D004800:Enzymes, Immobilized; D004912:Erythrocytes / Q000201:enzymology; D010088:Oxidoreductases; D013154:Spleen / Q000201:enzymology; D013194:Staining and Labeling; D013379:Substrate Specificity
D004798:Enzymes; D004800:Enzymes, Immobilized; D010088:Oxidoreductases