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 initiation. Two families of transcription factors, NFkappaB 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 NFkappaB (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.
Rosenau
Christopher
C
Perbio Science Bioresearch Division, Pierce Biotechnology Inc., 2202 North Bartlett Avenue, Milwaukee, WI 53202, USA. christopher.rosenau@piercenet.com
Emery
Daryl
D
Kaboord
Barbara
B
Qoronfleh
M Walid
MW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA