TransAM Flexi NFκB Kits provide everything needed to study Nuclear Factor κB (NFkB), including a positive control extract and positive control biotinylated oligonucelotide. The TransAM Flexi format enables you to look at NFκB binding sites of your own design. This kit works by combining a stimulated nuclear extract with biotinylated oligos you generated followed by transfer to a 96-well streptavidin coated plate to capture activated NFκB binding. Kits are available with antibodies specific for the activated form of p50 and p65. A family kit is also available with the ability to screen the members of the NFkB family (p50, p52, p65, c-Rel and RelB). Recombinant p50 and Recombinant p65 proteins are available separately to generate an optional protein standard curve in the TransAM NFκB Transcription Factor ELISA kits. The TransAM® Flexi tab below provides complete method details; the NFkB Info tab gives an overview of the NFκB transcription factor as well as kit data; the kit manual can be downloaded under the Documents tab.
Measuring NFκB p65 at different binding sites.
|TransAM® Flexi NFκB Family||2 x 96 rxns||43298||$1,250||Buy|
|TransAM® Flexi NFκB p50||1 x 96 rxns||41098||$740||Buy|
|TransAM® Flexi NFκB p65||1 x 96 rxns||40098||$740||Buy|
|TransAM® NFκB Flexi Family Manual|
|TransAM® NFκB Flexi Manual|
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|Tools for Disease Research|
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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 NFκB
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.
Contents & Storage
TransAM Flexi Kits are designed for customers interested in studying alternative binding sites of NFκB 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 NFκB Kits, is provided to perform 40 control reactions. If you are interested in looking at only the consensus site of NFκB, please use the original TransAM NFκB Kits, as they conveniently provide the oligo precoated to the plate. Flexi Kits also include one or two 96-well streptavidin-coated assay plate(s) with plate sealer(s), NFκB p50 or p65 or p50, p52, p65, c-Rel and RelB primary antibody(ies), HRP-conjugated secondary antibody, NFκB wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Herring Sperm DNA, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing 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.
NOTE: the p50 and p52 antibodies can be used with human and mouse extracts, while the p65 and RelB antibodies can be used with human, mouse and rat extracts. The c-Rel antibody can only be used with human extracts.
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