Enzymoblotting: Visualization of electrophoretically separated enzymes on nitrocellulose membranes using specific substrates

Enzymoblotting is a technique for the detection of electrophoretically separated enzymes after transfer to nitrocellulose (NC) membranes and subsequent incubation with suitable substrates. After agarose gel electrophoresis or isoelectric focusing in ultrathin polyacrylamide gels, blotting of the separated proteins/enzymes to NC membranes was performed by capillary forces for 30 min. Hydrolases, e. g. pancreatic proteinases, were visualized using amino acid/peptide substrates based either on the β‐naphthol or para‐nitroanilide (pNA) group. Both types of substrates were used with good results, but the pNA substrates provide greater specificity and sensitivity. An oxidoreductase, lactate dehydrogenase, was visualized using an electron transfer dye staining method. Enzymoblotting also allows detection of inactive proenzymes since conversion to active enzymes in situ on the NC membrane may be performed by pre‐incubating the NC membranes with activation enzymes. The enzymoblotting technique offers several advantages over traditional gel staining methods. The enzymes are immobilized on the NC membrane, preventing diffusion during substrate incubation, and as a result, high resolution is retained even after long incubation times; the product obtained as a result of the enzyme activity can be chemically derivatized and bound to the membrane, thus widening the categories of possible substrates; subsequent incubation of the same NC membrane in various substrate solutions can be performed; enzymoblotting can be used in combination with other techniques, e. g., immunoblotting for parallel structural and functional studies.