The product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels.
A method termed "product-selective" blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase (EC 3.5.1.2) or glutamate synthase (EC 1.4.7.1) reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing "image gel." Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.
D002852:Chromatography, Ion Exchange; D004587:Electrophoresis, Agar Gel; D004592:Electrophoresis, Starch Gel; D005970:Glutamate Synthase / Q000032:analysis*; D005972:Glutaminase / Q000032:analysis*; D011874:Radiometry; D013050:Spectrometry, Fluorescence; D000637:Transaminases / Q000032:analysis*
D005970:Glutamate Synthase; D000637:Transaminases; D005972:Glutaminase