Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein com- plexes binding to DNA with varied specificity and affinity. Our approach is based on the novel cou- pling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double- stranded DNA microarrays and dye-labeled regula- tory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experi- ment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF-PBM are similar to those determined by traditional methods, while simultaneous measure- ment of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF-PBM provides a novel and extendible platform for multi-protein transcription factor investigation.