Detection of Enzymes by Substrate Staining

Activity stains are of great importance during the isolation, purification, and characterization of enzymes, since a particular catalytic reaction is involved and the detection of this activity leads to the unequivocal identification of the zone of interest on the electrophoresis gel. Following separation, the gel is removed from the electrophoresis apparatus and is immersed in a minimal volume of a substrate solution. Detection relies on the formation of a colored product by enzyme in the zones containing the enzyme. Examples of activity stains are given in Table 9.2. 

Red cell acid phosphatase Phosphoglucomutase Placental alkaline phosphatase 

Succinate, P-hydroxybutyric and glutamate dehydrogenase Catalase Caeruloplasmin Leucine aminopeptidase 

Phenolphthalein diphosphate followed by alkali Reduction of the tetrazolium salt of MTT P-Naphthyl phosphate and diazo salt of fast blue RR 

8-Hydroxyquinoline plus blue RR salt 6-Bromo-2-naphthylcarbonaphthoxycholine iodide plus blue B salt Application of agar overlay containing a tetrazolium salt 

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