The TransAM® Flexi 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® Flexi 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 450nm
- 96-stripwell format enables both high and low throughput
TransAM Flexi Kit applications include:
- Study of variant transcription factor binding sites
- Analysis of native promoters
- Confirmation of chromatin immunoprecipitation (ChIP) results
- Determination of isoform-binding affinity for NF?B
TransAM® Flexi transcription factor ELISA Kits
TransAM Flexi Kits are perfect for researchers interested in studying alternative binding sites for their transcription factor. In TransAM Flexi, the researcher synthesizes a biotinylated oligo or PCR product, which contains the transcription factor-binding site of choice. A stimulated nuclear extract is mixed with this biotinylated oligo and then transferred to the wells of a 96-well streptavidin coated plate, where the biotinylated oligo containing the activated transcription factor is captured. Next, a primary antibody specific for an epitope on the bound and active form of the transcription factor is 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 Flexi process.
* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NF?B-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).
Contents & Storage
TransAM Flexi Kits are designed for customers interested in studying alternative binding sites of NF?B and require you to design and synthesize your own oligos of interest, which will then be coated onto the plate. Enough control biotinylated oligo, which contains the consensus-binding site used in the original TransAM NF?B Kits, is provided to perform 40 control reactions. If you are interested in looking at only the consensus site of NF?B, please use the original TransAM NF?B Kits, as they conveniently provide the oligo precoated to the plate. Flexi Kits also include one or two 96-we