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Journal of Biomolecular Screening
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Development of a High-Throughput Plate-Based Chemiluminescent Transcription Factor Assay

Christopher Rosenau

Perbio Science Bioresearch Division - Pierce Biotechnology, Inc.

Daryl Emery

Perbio Science Bioresearch Division - Pierce Biotechnology, Inc.

Barbara Kaboord

Perbio Science Bioresearch Division - Pierce Biotechnology, Inc.

M. Walid Qoronfleh

Perbio Science Bioresearch Division - Pierce Biotechnology, Inc.

Transcription factors are DNA-binding proteins that regulate the expression of specific genes by controlling transcription initiation. Two families of transcription factors, NF{kappa}B 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 NF{kappa}B (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 assay 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.

Key Words: transcription factors • chemiluminescence • electrophoretic mobility shift assay (EMSA) • nuclear factor kappa B (NF{kappa}B) • activator protein-1 (AP-1) • DNA-binding proteins • gel shift assay

Journal of Biomolecular Screening, Vol. 9, No. 4, 334-342 (2004)
DOI: 10.1177/1087057103261446
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