TransAM® NFAT Transcription Factor ELISA Kits
TransAM NFAT Kits provide everything needed to study Nuclear Factor of Activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), including a positive control extract. The kit can be used with human and mouse extracts. See the NFAT Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.
|TransAM® NFATc1 Manual|
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NFAT Transcription Factor Info
The Nuclear Factor of Activated T cells (NFAT) family of transcription factors contains five members (NFAT 1-5). The NFATC transcription complex consists of two components: a preexisitng cytosolic component that translocates to the nucleus and an inducible nuclear component. NFATc1, also known as NFATC or NFAT2, has multiple isoforms as a result of alternative splicing, that regulate inducible expression of cytokine genes in T-cells. NFATc1 is confined to the cytoplasm in resting cells, where it maintains its phosphorylation state by the action of constitutive kinases. Upon stimulation, NFATc1 is dephosphorylated by calcineurin and migrates to the nucleus. NFATc1 can be stimulated by T cell receptor (TCR) stimulation and calcium ionophores. Cyclosporin A and FK506 immunosuppressor drugs inhibit calcineurin activity on NFAT.
Figure 1: Monitoring NFATc1 activation with the TransAM NFATc1 Kit.
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.
Why use TransAM® transcription factor ELISAs?
- 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 at 450 nm
- 96-stripwell format enables both high and low throughput
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).
Figure 1: Flow chart of the TransAM process.
Contents & Storage
One or five NFAT 96-well assay plate(s) with plate sealer(s), NFATc1 primary antibody, HRP-conjugated secondary antibody, NFAT wild-type and mutated oligonucleotides, Jurkat nuclear extract (positive control), Dithiothreitol, Protease Inhibitor Cocktail, poly[d(I-C)], Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing and Stop Solutions. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.
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