Abstract
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.
MeSH terms
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Activating Transcription Factor 2
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Animals
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Antibody Specificity
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Base Sequence
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Cell Extracts
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Cell Nucleus
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Consensus Sequence
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Cyclic AMP Response Element-Binding Protein / genetics
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Cyclic AMP Response Element-Binding Protein / immunology
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Cyclic AMP Response Element-Binding Protein / metabolism
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DNA / genetics
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DNA / metabolism
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HeLa Cells
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Humans
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Immunoenzyme Techniques / methods*
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Jurkat Cells
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Mammals
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NF-kappa B / genetics
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NF-kappa B / immunology
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NF-kappa B / metabolism
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NF-kappa B p50 Subunit
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Proto-Oncogene Proteins c-fos / genetics
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Proto-Oncogene Proteins c-fos / immunology
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Proto-Oncogene Proteins c-fos / metabolism
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Sensitivity and Specificity
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Transcription Factors / genetics*
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Transcription Factors / immunology
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Transcription Factors / metabolism
Substances
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ATF2 protein, human
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Activating Transcription Factor 2
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Cell Extracts
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Cyclic AMP Response Element-Binding Protein
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NF-kappa B
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NF-kappa B p50 Subunit
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Proto-Oncogene Proteins c-fos
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Transcription Factors
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DNA