10.1177/1087057103261446Rosenau et al.Chemil minescent Transcription Factor Assay Development of a High-Throughput Plate-Based Chemiluminescent Transcription Factor Assay

Transcription factors are DNA-binding proteins that regulate the expression of specific genes by controlling transcription ini-tiation. Two families of transcription factors, NFκB and AP-1, play pivotal roles in controlling important cellular processes ranging from normal cell growth and differentiation to apoptosis and cancer. Identifying changes in the DNA-binding activity of these factors is essential to understanding the regulation of these processes. We have developed a high-throughput DNA-based ELISA capable of monitoring activated levels of NFκB (p50 and p65) and AP-1 (c-Jun and c-Fos). This chemiluminescent assay utilizes a 96-well plate format, eliminating the throughput challenges imposed by traditional gel shift assays and exceeding the sensitivity and dynamic range of standard colorimetric detection systems. The sensitivity of this as-say enables distinction between subtle as well as dramatic differences in the DNA-binding activity of these factors that result from the treatment of cells with various inhibitors or activating agents. (Journal ofBiomolecularScreening2004:334-342) Key words: transcription factors, chemiluminescence, electrophoretic mobility shift assay (EMSA), nuclear factor kappa B (NFκB), activator protein-1 (AP-1), DNA-binding proteins, gel shift assa