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TransAM® ATF-2

dna-binding ELISA for activated ATF-2 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® ATF-2 Transcription Factor ELISA Kits

TransAM ATF-2 Kits provide everything needed to study Activating Transcription Factor 2 (ATF-2) phosphorylated at threonine 71, including a positive control extract. The kit can be used with human extracts. Reactivity with other species has not been determined. Because of ATF-2's regulation by MAPK, we also offer the TransAM MAPK Family Kit for studying the MAPK regulated transcription factors ATF-2, c-Jun, c-Myc, MEF2 and STAT1α. See the ATF 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® ATF-2 1 x 96 rxns 42396 $665 Buy Now
5 x 96 rxns 42896 $2,795 Buy Now

ATF-2 Transcription Factor Info

ATF-2 (activating transcription factor 2, also known as CREB2 or cAMP responsive element binding protein 2) is a member of the ATF/CREB transcription factor family that binds to the cAMP response element (CRE). ATF-2 belongs to a leucine zipper family of DNA binding proteins and forms homodimers or heterodimers with c-Jun to stimulate CRE-dependent transcription. ATF-2 is activated by three MAPK pathways, ERK1/2, JNK and p38. Phosphorylation of Thr71 by MAPK14 or JNK increases transcriptional activity. TransAM ATF-2 Kits contain an antibody specific for the phosphorylated form of ATF-2 (Thr71) when bound to its target DNA.

Active Motif also offers our FACE™ In-cell Western Phospho ELISAs for quantifying and comparing the phosphorylated and total amounts of ERK1/2, JNK and p38 proteins.

 
 
The TransAM ATF-2 Kit was used to test increasing amounts of nuclear extract from unstimulated HeLa and HL-60 cells for activated AML-1/Runx1
 
Figure 1: Monitoring ATF-2 activation with the TransAM ATF-2 Kit.

Increasing amounts of nuclear extract from unstimulated HeLa and Anisomycin-stimulated HeLa cells are tested for ATF-2 activation using the TransAM ATF-2 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

One or five ATF 96-well plate(s) with plate sealer(s), primary antibody, HRP-conjugated secondary antibody, wild-type and mutated competitor oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, 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.