ENZYME AND ANTIBODY DETECTION FOLLOWING ISOELECTRIC FOCUSING ON ULTRATHIN-LAYER REHYDRATED POLYACRYLAMIDE GELS

Abstract

Rehydratable polyacrylamide and conventionally prepared ultrathin-layer gels (250-375μm thick) were compared. A desired ampholyte, substrate and dye, or antibody was introduced into the dried gels by rehydration. Conventional gels were used both fresh or after storage in sealed aluminized bags. The lower conductance in rehydrated gels, as compared to conventionally cast gels, allowed higher voltage gradients to be applied at controlled Joule heat loads, with increased resolution of the system. Studies with erythrocyte acid phosphate (EAP) indicated that increased activity and greater resolution were obtained in the rehydrated gels as opposed to those cast in the conventional manner. In addition, glycerol also was able to reduce conductance further, however, glycerol also reduced enzyme activity of erythrocyte acid phosphatase (EAP) C which may be caused by an isomeric conversion to EAP B in its presence. Impregnation of antibody into the gel by rehydration produced a material for immuno-sandwich overlays that allowed reaction times of as little as three minutes with focused Gc proteins to produce a specific precipitin. Unreacted antibody was washed completely from the gel in as little as 30min leaving sharply resolved precipitin lines in the gel that could be stained with both Coomassie Brilliant Blue R250 or with silver techniques. Studies of the isoelectric points by automated laser microdensitometry indicated a high degree of reproducibility between gels.