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.
|TransAM® ATF-2||1 x 96 rxns||42396||$665||Buy Now|
|5 x 96 rxns||42896||$2,795||Buy Now|
|TransAM® ATF-2 Manual|
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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.
Figure 1: Monitoring ATF-2 activation with 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).
Figure 1: Flow chart of the TransAM process.
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.