Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
- Sensitivity: 600 ng/well
Product nameSTAT (STAT1alpha, STAT3, STAT5A, STAT5B) Transcription Factor Assay Kit (Colorimetric)
Sample typeNuclear Extracts
Sensitivity< 600 ng/well
Assay time3h 30m
Species reactivityReacts with: Mouse, Rat, Human
The STAT1α, STAT5A and STAT5B antibodies can be used with human and rat extracts, while the STAT3 antibodies can be used with human, mouse and rat extracts.
STAT (STAT1alpha, STAT3, STAT5A, STAT5B) Transcription Factor Assay Kit (Colorimetric) (ab207228) is a high throughput assay to quantify activation of STAT factors (STAT1alpha, STAT3, STAT5A, STAT5B) at the same time in one plate. 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 STAT (STAT1alpha, STAT3, STAT5A, STAT5B) consensus binding site (5’ – TTCCCGGAA – 3’) has been immobilized onto a 96-well plate. Active STAT (STAT1alpha, STAT3, STAT5A, STAT5B) present in the nuclear extract specifically binds to the oligonucleotide. STAT (STAT1alpha, STAT3, STAT5A, STAT5B) is detected by a primary antibody that recognizes an epitope of STAT (STAT1alpha, STAT3, STAT5A, STAT5B) 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 and rat STAT (STAT1alpha, STAT3, STAT5A, STAT5B). STAT3 can also be detected in mouse.
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 non-covalently 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 de-phosphorylation 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.
Storage instructionsPlease refer to protocols.
Components 2 x 96 tests 10X Antibody Binding Buffer 2 x 2.2ml 10X Wash Buffer 1 x 60ml 96-well IRF-3 assay plate 2 units Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 2 x 11µl Binding Buffer 1 x 10ml Developing Solution 2 x 11ml Dithiothreitol (DTT) (1 M) 1 x 100µl Herring sperm DNA (1 μg/μL) 1 x 100µl Lysis Buffer 1 x 10ml Nb nuclear extract (prolactin stimulated) (2.5 μg/μL) 1 x 40µl Plate sealer 2 units Protease Inhibitor Cocktail 1 x 100µl STAT mutated oligonucleotide (10 pmol/μL) 1 x 100µl STAT Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl STAT1α antibody 1 x 11µl STAT3 antibody 1 x 20µl STAT5A antibody 1 x 11µl STAT5B antibody 1 x 11µl Stop Solution 1 x 60ml
Cellular localizationSTAT3: Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm. Constitutive nuclear presence is independent of tyrosine phosphorylation. Predominantly present in the cytoplasm without stimuli. Upon leukemia inhibitory factor (LIF) stimulation, accumulates in the nucleus. The complex composed of BART and ARL2 plays an important role in the nuclear translocation and retention of STAT3. STAT 5A + B: Cytoplasm. Nucleus. Translocated into the nucleus in response to phosphorylation.
Nuclear extracts from Nb2 cells stimulated with prolactin) were assayed for activity of STAT family members: STAT1alpha, STAT3, STAT5A, STAT5B in the absence (grey) or presence of wild-type (black) or mutated (white) consensus binding oligonucleotides. Note that the wild-type oligonucleotide reduces STAT binding by over 90% while incubation with the mutant STAT competitor oligo has limited effect on STAT binding to DNA. These results are provided for demonstration purposes only.
ab207228 has not yet been referenced specifically in any publications.