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Abstract for Novel cAMP Assays Using A Genetically Encoded
Luciferase Biosensor
Frank Fan, Brock Binkowski, Braeden Butler, Pete Stecha and Keith
Wood
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
Preferred methods for cell-based GPCR assays involve direct
detection of intracellular signal transduction events. Among the
most successful are methods using fluorescent dyes or aequorin for
real-time monitoring of intracellular calcium. However, analogous
technologies have been lacking for the detection of intracellular
cAMP dynamics. We have recently created a novel chimeric molecule by
combining circularly permuted firefly luciferase with the allosteric
RIIβB cAMP binding domain of Protein Kinase A to create a sensor
capable of emitting luminescence in proportion to the concentration
of cAMP. The sensor design was based on earlier results showing that
cleavage of protease sensors using circularly permuted luciferase
could greatly activate luminescence. Live cell, zero-step GPCR
assays using this sensor allow the dynamic detection of changes in
cAMP concentration using stable or transiently transfected cell
lines. In addition, it is possible to develop a
single-step homogenous assay format for detection of cAMP in vitro. |
