Coupling of membrane-bound and detergent-solubilized sodium- and potassium-activated ATPase to p-benzoquinone-treated microtiter plates.
Membrane-bound and dodecyloctaoxyethyleneglycol monoether-solubilized Na,K-ATPases from pig kidney were covalently attached to microtiter plate wells pretreated with p-benzoquinone (plus collodion for some plates). The immobilized enzymes were detected with the mouse monoclonal antibody (named 38) specific to Na,K-ATPase and a perioxidase-conjugated rabbit IgG anti-mouse IgG. When the two Na,K-ATPase preparations were applied to each well at the same protein concentration, the color intensity of the peroxidase reaction for determination of antibody was two to three times stronger with the solubilized enzyme than with the membrane-bound enzyme. Similar titer values were obtained from the graphical analysis of titration curves of both enzymes. Red cell membrane proteins as well as Na,K-ATPase were covalently attached to the plastic. p-Benzoquinone should be generally useful for coupling membrane proteins, even in detergent solutions, to microtiter plate wells.
D000818:Animals; D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D003902:Detergents; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D007668:Kidney / Q000201:enzymology; D011506:Proteins; D011809:Quinones; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*; D012995:Solubility; D013552:Swine
D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D003902:Detergents; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D011506:Proteins; D011809:Quinones; C004532:quinone; D000254:Sodium-Potassium-Exchanging ATPase