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Gene Regulation

TransAM® STAT3 & STAT Family Kits

dna-binding ELISAs for activated STAT1α, STAT3, STAT5A and STAT5B

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® STAT Transcription Factor ELISA Kits

TransAM STAT Kits provide everything needed to study Signal Transducers and Activators of Transcription (STAT), including a positive control extract. TransAM STAT3 Kits are available with antibodies specific for the activated form of STAT3. A TransAM STAT Family kit is also available with the ability to independently screen the members of the STAT family (STAT1α, STAT3, STAT5A & STAT5B) simultaneously. The STAT1α, 5A and 5B antibodies can be used with human and rat extracts, while the STAT3 antibodies can be used with human, mouse and rat extracts. See the STAT 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® STAT Family 2 x 96 rxns 42296 $1,125 Buy Now
TransAM® STAT3 1 x 96 rxns 45196 $665 Buy Now
5 x 96 rxns 45696 $2,795 Buy Now

STAT Transcription Factor Info

Signal Transducers and Activators of Transcription (STAT) are a family of latent cytoplasmic proteins that are activated in response to cytokines and growth factors. The STAT family has seven members: STAT1, 2, 3, 4, 5A, 5B and 6. STATs are involved in many pathways that regulate cellular functions in the lympho-hematopoietic system. STATs can be activated by ligands such as interferons (IFNs), epidermal growth factor (EGF), platelet derived growth factor (PDGF) and interleukins (IL). When members of the STAT family are phosphorylated they form homo- or heterodimers and translocate to the nucleus to activate transcription. STAT1α homodimerizes upon tyrosine or serine phosphorylation induced by IFNγ, but forms heterodimers with STAT2 when induced by IFNα/β. STAT3 is activated by phopshorylation of tyrosine 705 and serine 727 to mediate the expression of genes involved in cell growth and apoptosis. STAT5A and STAT5B are activated in response to several cell ligands and growth hormones. They are capable of serving as both transcriptional activators and signal transducers.


Figure 1: Monitoring STAT Family member activation using the TransAM STAT Family Kit.

STAT1α, STAT3, STAT5A and STAT5B activation were assayed using the TransAM STAT Family Kit. 1:1000 dilutions of each antibody in the kit were tested using 5 µg/well of nuclear extract prepared from a stimulated cell line: STAT1α was tested with U-937 (TPA + IFNγ), STAT3 with Hep G2 (IL-6, 100 ng/ml), and STAT5A and STAT5B with Nb2 (prolactin). Assays were performed in the absence or presence of 20 pmol of competitor oligonucleotide that contains either a wild-type or mutated STAT consensus binding site. Note that the wild-type oligonucleotide reduces STAT binding by over 90%, while incubation with the mutant STAT competitor oligo has a limited effect on STAT 1α, STAT3, STAT5A or STAT5B binding to DNA.

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, two or five STAT 96-well assay plate(s) with plate sealer(s), STAT3 or STAT1α, STAT3, STAT5A and STAT5B primary antibodies, HRP-conjugated secondary antibody, STAT wild-type and mutated 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.