Multiple transcription factor profiling by enzyme-linked immunoassay.
Transcription factor-DNA interactions have been widely studied in the regulation of gene expression. We have established an enzyme-linked immunoassay platform to quantify specific transcriptionfactor-DNA interactions. In our assay, dsDNA immobilized on a 96-well plate captures the transcriptionfactor from the nuclear extract of mammalian cells. The DNA-bound transcription factor is detected and quantified by enzyme-linked immunoassay using a transcription factor-specific antibody. We have profiled multiple transcription factors involved in inflammation including NFkappaB p50, NFkappaB p65, c-Rel, c-Fos, CREB-1, and ATF-2. When compared with the traditional electrophoretic mobility shift assay, the enzyme-linked immunoassay shows a 10-fold higher sensitivity, eliminates the use of radioactivity, allows for a high-throughput format, and is faster.
Shen
Z
Z
BD Biosciences Clontech, Palo Alto, CA 94303, USA.
Peedikayil
J
J
Olson
G K
GK
Siebert
P D
PD
Fang
Y
Y
D051698:Activating Transcription Factor 2; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002457:Cell Extracts; D002467:Cell Nucleus; D016384:Consensus Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D019169:Jurkat Cells; D008322:Mammals; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D002457:Cell Extracts; D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D016760:Proto-Oncogene Proteins c-fos; D014157:Transcription Factors; D004247:DNA