STAT3 Transcription Factor Assay Kit (Colorimetric)

STAT3 Transcription Factor Assay Kit (Colorimetric) (ab207229) is a high throughput assay to quantify STAT3 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.

A specific double stranded DNA sequence containing the STAT3 consensus binding site (5' – TTCCCGGAA – 3') has been immobilized onto a 96-well plate. Active STAT3 present in the nuclear extract specifically binds to the oligonucleotide. STAT3 is detected by a primary antibody that recognizes an epitope of STAT3 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human, mouse and rat STAT3.

Key performance and benefits:

• Assay time: 3.5 hours (cell extracts preparation not included).
• Detection limit: < 0.6 μg nuclear extract/well.
• Detection range: 0.3 – 10 μg nuclear extract/well.

STAT (signal transducers and activators of transcription) transcription factors were discovered fourteen years ago as mediators of interferon-induced gene expression. They comprise a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Their critical role in development and normal cell signaling has been largely determined through the analysis of transgenic mice lacking individual STAT genes. The STAT family consists of seven members that are activated by virtually every cytokine and growth factor.

The STAT proteins are unique among transcription factors in containing an SH2 (src-homology 2), phosphotyrosine-binding domain, a common protein-protein interaction domain among signaling proteins. Tyrosine phosphorylation around residue 700 is essential for the dimerization of STATs and the concomitant nuclear translocation of the dimer. Ligand-activated receptors that catalyze this phosphorylation include receptors with intrinsic tyrosine kinase activity (epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony-stimulating factor-1) as well as receptors that lack intrinsic tyrosine kinase activity but to which Janus kinases (JAKs) are noncovalently associated. Receptors to which JAKs are bound are often referred to as cytokine receptors. Their ligands include IFN-α, -β and -γ; interleukins (IL) 2 to 7, 10 to 13, and 15; and erythropoietin, growth hormone, prolactin, thrombopoietin and other polypeptides. STAT dimers and heterodimers, but not monomers, are competent to bind DNA. The known DNA binding heterodimers are STAT1:2 (strong binding requires the joint presence of another protein, p48) and STAT1:3. STATs that form homodimers that bind DNA include STAT 1, 3, 4, 5 (STAT5A and 5B interact in a manner equivalent to a heterodimer) and 6.

In most cases, STAT activation is transient. Inactivation of STAT proteins is carried out by several mechanisms, including dephosphorylation of STAT proteins in the nucleus and degradation through the ubiquitin-proteosome pathway. A novel family of negative feedback inhibitors of the JAK-STAT pathway has been identified, referred to as suppressor-of-cytokine-signaling (SOCS) proteins/JAK binding (JAB) proteins, and STAT-induced STAT inhibitors (SSIs). In addition, a family of protein inhibitors of activated STAT (PIAS) proteins has been identified.