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TransAM® PPARγ

dna-binding ELISA for activated PPARγ transcription factor

TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.

TransAM® PPARγ Transcription Factor ELISA Kits

TransAM PPARγ Kits provide everything needed to study Peroxisome Proliferator-Activated Receptor gamma (PPARg), including a positive control extract. The kit can be used to detect gamma 1 and gamma 2 from human, mouse and rat extracts. It will not cross-react with alpha or delta isotypes. See the PPAR Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
TransAM® PPARγ 1 x 96 rxns 40196 $665 Buy Now
5 x 96 rxns 40696 $2,795 Buy Now

PPAR Info

Peroxisome Proliferator-Activated Receptors (PPARs) are nuclear receptors involved in lipid transport and metabolism. As such, their roles in chronic diseases such as diabetes, obesity, atherosclerosis and cancer are heavily investigated. Transcriptional activity of PPARs is regulated by fatty acid binding. Three PPAR isotypes have been identified: α, δ and γ. PPARγ stimulates lipolysis of circulating triglycerides and the subsequent uptake of fatty acids into adipose cells. PPARs bind to peroxisome proliferator response elements (PPREs) as heterodimers with the retinoid X receptor (RXR).

 

  

Figure 1: Monitoring PPAR activation with the TransAM PPARγ Kit.

Different amounts of nuclear extracts from unstimulated (opened squares) and PMA-stimulated (filled circles) THP-1 cells are tested for PPAR activation using the TransAM PPARγ 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).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* 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).

Contents & Storage

Each TransAM PPARγ Kit contains PPARγ primary antibody, HRP-conjugated secondary antibody, PPARγ wild-type and mutated oligonucleotides, positive control nuclear extract, Dithiothreitol, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis Buffer, Binding Buffer, 10X Washing Buffer, 10X Antibody Binding Buffer, Developing Solution, Stop Solution and one or five PPARγ 96-well assay plate(s) with plate sealer(s). 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.