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AP-1 proteins play a role in the expression of many genes involved in cellular proliferation and cell cycle progression. The transcription factor AP-1 is composed of a mixture of heterodimeric protein complexes derived from the Fos and Jun families, including c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD. AP-1 heterodimers bind to DNA on a serum response element with the 5´-TGA(C/G)TCA-3´ sequence. Jun proteins can bind to form transcriptionally active homodimers or heterodimers with CREB/ATF members to bind the CRE element. AP-1 expression is induced by multiple stimuli, including the presence of serum, growth factors, phorbol esters and oncogenes. Phosphorylation of AP-1 family members by kinases is required for transactivation activity.
The TransAM™ 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.
Why use TransAM?
- Up to 100-fold more sensitive than gelshift assays
- Eliminates the use of radioactivity and the need to run gels
- Results in less than five hours
- Colorimetric readout enables easy, quantitative analysis with spectrophotometry
- 96-stripwell format enables both high and low throughput
How TransAM™ Kits 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 or chemiluminescent readout (Figure 1).
Figure 1: Flow chart of the TransAM process.
* 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).
