TransAM® Flexi NF?B p50

by Active Motif
Login to view price.

Description

TransAM Flexi NF?B Kits provide everything needed to study Nuclear Factor ?B (NFkB), including a positive control extract and positive control biotinylated oligonucelotide. The TransAM Flexi format enables you to look at NF?B 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 NF?B 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 NF?B Transcription Factor ELISA kits. The TransAM® Flexi tab below provides complete method details; the NFkB Info tab gives an overview of the NF?B transcription factor as well as kit data; the kit manual can be downloaded under the Documents tab.

 TransAM NFkB Flexi data

Measuring NF?B p65 at different binding sites.

Five µg of nuclear extract from untreated and TNF-?-treated HeLa cells were used to assay the binding affinity of NF?B p65 for four different biotinylated 50-mer oligos. Each of the 3 test oligos (Ig?, IL-8 and uPA) contained the wild-type binding site of a promoter regulated by NF?B. These sites were compared to the consensus-binding site control provided in TransAM Flexi NF?B Kits.

 

 

 

* 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).

 
Name Format Cat No. Price  
TransAM® Flexi NF?B Family 2 x 96 rxns 43298 $1,145 Buy Now
TransAM® Flexi NF?B p50 1 x 96 rxns 41098 $685 Buy Now
TransAM® Flexi NF?B p65 1 x 96 rxns 40098 $685 Buy Now

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).

TransAM Flexi flow chart
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

You recently viewed

Clear recently viewed