LUMINESCENCE DETECTION REAGENTS FOR PROTEINS

ELA TECHNOLOGIES, INC., HAS PRODUCED TWO 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 OFITS SUBSTRATE, COELENTERATE LUCIFERIN. THIS RESEARCH 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: (1) TO USE BIOTINYLATED PROTEINS ON NITROCELLULOSE AND NYLON BLOTS AFTER TRANSFER FROM SLAB GEL ELECTROPHORETOGRAMS, (2) TO USE BIOTINYLATED RECAEQUORIN FORDETECTION 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 1 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.