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基因调控

TransAM® 转录因子活性检测ELISAs

检测转录因子活性的灵敏的,可定量的ELISA

Transcription factors are DNA-binding proteins that tightly regulate gene expression. They consist of two distinct domains – one that displays high affinity for a specific DNA sequence and one that confers transcriptional activity. Transcription factors are activated by phosphorylation of specific residues or by processing bound inhibitory proteins. Understanding and quantifying transcription factors is essential for the study of cell functions in relation to differentiation, brain activity, immune response, inflammation and various disease states.

TransAM® Kits are sensitive, non-radioactive transcription factor ELISA kits that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets; choose a kit from the right column, then choose the TransAM® Method tab at the bottom of the page for complete assay details.

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

 

The TransAM® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

How TransAM® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).