ELA Technologies, Inc. has produced and will sell recombinant, bioluminescent proteins designated RecAequorin and RecLuciferase. These proteins produce light with high efficiency and are detectable on film in the picogram range. RecAequorin produces a flash of light upon the addition of calcium and RecLuciferase produces a sustained signal in the presence of its substrate, coelenterate luciferin. The research proposed will evaluate the utility of these recombinant proteins as tags for the detection of biotinylated proteins and protein antigens on nitrocellulose and nylon membranes using both instant and X-ray film. This will be accomplished by completion of the following specific aims: l) to use biotinylated proteins on nitrocellulose and nylon """"""""blots"""""""" after transfer from slab gel electrophoretograms; 2) to use biotinylated RecAequorin for detection of biotinylated second antibodies in Western """"""""blot"""""""" techniques; 3) to prepare biotinylated RecLuciferase and evaluate the usefulness of its sustained signal for applications described in Specific Aims l and 2; and 4) to prepare streptavidin conjugates of both RecAequorin and RecLuciferase as direct substitutes for the commonly used enzyme conjugates such as avidin/alkaline phosphatase. Direct comparisons will be made between the recombinant bioluminescent protein tags and the enzyme derivatives and conjugates for protein detection on nylon and nitrocellulose membranes.
|Stults, N L; Stocks, N F; Rivera, H et al. (1992) Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli. Biochemistry 31:1433-42|