Enzyme product blot for nondestructive assay of protein catalytic function in polyacrylamide gels.
A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase.
Minter
M
M
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Towbin
J
J
Harter
J
J
McCabe
E R
ER
D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D005991:Glycerol Kinase; D006593:Hexokinase; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D011506:Proteins; D006593:Hexokinase; D005991:Glycerol Kinase