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 nameGATA (GATA1, GATA2, GATA3) Transcription Factor Assay Kit (Colorimetric)
Sample typeNuclear Extracts
Sensitivity< 600 ng/well
Assay time3h 30m
Species reactivityReacts with: Human
GATA (GATA1, GATA2, GATA3) Transcription Factor Assay Kit (Colorimetric) (ab207205) is a high throughput assay to quantify activation of GATA family members at the same time in one assay. 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 GATA consensus binding site (5’ –AGATAA– 3’) has been immobilized onto a 96-well plate. Active GATA present in the nuclear extract specifically binds to the oligonucleotide. GATA is detected by a primary antibody that recognizes an epitope of GATA accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout at OD 450 nm. This product detects only human GATA.
Key performance and benefits:
- Assay time: 3.5 hours (cell extracts preparation not included).
- Detection limit: < 0.6 µg nuclear extract/well.
- Detection range: 0.6 – 5 µg for GATA1 and GATA2; 0.6 – 10 µg for GATA3 nuclear extract/well.
The GATA family of transcription factors consists of 6 members: GATA 1-6. The family is defined by the presence of 1 or 2 zinc fingers containing four cysteine residues with the motif CX2CX12CX2C that coordinate a single zinc ion. In mammals, the N-finger is important for interactions with cofactors such as Friend of GATA (FOG), while the C-finger binds to (A/T)GATA(A/G) motifs. Genes encoding GATA factors are transcriptionally regulated in a tissue-restricted manner. They include platelet factor 4 (PF4), glycoprotein (GP) IIB, α-myosin heavy chain, thyroid transcription factor-1 (TTF-1) and H+/K+-ATPase. The members of the GATA family have been grouped into two subfamilies: GATA-1, -2 and -3 are expressed mainly in hematopoietic cells, and GATA-4, -5 and -6 are found predominantly in the heart and gut.
GATA-1, the founding member of the family is essential for normal erythropoiesis and megakaryocyte differentiation. It is expressed in erythroid cells, megakaryocytes, mast cells, eosinophils and testis. Thought to be a negative regulator of cell proliferation in early megakaryocyte progenitors, GATA-1 has also been implicated in regulation of terminal differentiation markers in various myeloid cells. Mutations in GATA-1 are associated with familial blood disorders. GATA-2 is expressed in vascular endothelial cells and embryonic brain and liver. It is thought to play a role in the regulation of endothelial gene expression, and is also found in megakaryocytes and mast cells with GATA-1. GATA-3 is found mainly in cells of T-cell lineage, and in low levels in mast cells. It is detected in the most immature subset of fetal day 12 thymocytes, and is essential for T-cell development.
GATA-4, -5 and -6 regulate gene expression in the heart, gut epithelium, liver, lung and gonad. Target genes of GATA-4, -5 and -6 include A- and B-type natriuretic peptides, cardiac actin and α-myosin heavy chain. GATA-4 is the transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. It can be found in the adult heart, ovary, testis, lung, liver and small intestine. During development, GATA-4 is expressed in the heart, gut, testis, ovary, liver, visceral endoderm and parietal endoderm. GATA-5 is expressed in the allantois, heart, lung bud, urogenital ridge, bladder and gut epithelium during development, and can be found in the adult small intestine, stomach, bladder and lungs. GATA-6 is thought to regulate the proliferation of cardiac progenitor cells. During development, it is expressed in the primitive streak, allantois, visceral endoderm, heart, lung buds, urigenital ridge, vascular smooth muscle cells and epithelial layer of the stomach, small intestine and large intestine. In adults, GATA-6 is found in the heart, aorta, stomach, small intestine and bladder.
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 GATA 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 GATA-1 antibody 1 x 11µl GATA-2 antibody 1 x 11µl GATA-3 antibody 1 x 11µl Herring sperm DNA (1 μg/μL) 1 x 100µl K-562 nuclear extract (5 μg/μL) 1 x 40µl Lysis Buffer 1 x 10ml Mutated oligonucleotide (10 pmol/μL) 1 x 100µl Plate sealer 2 units Protease Inhibitor Cocktail 1 x 100µl Stop Solution 1 x 60ml Wild-type oligonucleotide (10 pmol/μL) 1 x 100µl
FunctionTranscriptional activator or repressor which probably serves as a general switch factor for erythroid development. It binds to DNA sites with the consensus sequence 5'-[AT]GATA[AG]-3' within regulatory regions of globin genes and of other genes expressed in erythroid cells. Activates the transcription of genes involved in erythroid differentiation of K562 erythroleukemia cells, including HBB, HBG1/2, ALAS2 and HMBS (PubMed:24245781).
Involvement in diseaseX-linked dyserythropoietic anemia and thrombocytopenia
Thrombocytopenia with beta-thalassemia, X-linked
Anemia without thrombocytopenia, X-linked
Sequence similaritiesContains 2 GATA-type zinc fingers.
DomainThe two fingers are functionally distinct and cooperate to achieve specific, stable DNA binding. The first finger is necessary only for full specificity and stability of binding, whereas the second one is required for binding.
modificationsHighly phosphorylated on serine residues. Phosphorylation on Ser-310 is enhanced on erythroid differentiation. Phosphorylation on Ser-142 promotes sumoylation on Lys-137.
Sumoylation on Lys-137 is enhanced by phosphorylation on Ser-142 and by interaction with PIAS4. Sumoylation with SUMO1 has no effect on transcriptional activity.
Acetylated at 2 conserved lysine-rich motifs by CREBBP in vitro. Acetylation does not affect DNA-binding in vitro but is essential to induce erythroid differentiation and for binding chromatin in vivo (By similarity). Acetylated on Lys-233, Lys-245 Lys-246 by EP300.
- Information by UniProt
- Erythroid transcription factor
- GATA 2
Nuclear extracts (5 µg/well) were tested for GATA activation: K-562 cells were used for GATA-1 and GATA-2 detection and Jurkat cells were used for GATA-3 detection. Activation was monitored in absence (grey) and in the presence of wild-type (black) or mutated (white) consensus binding oligonucleotides. Note that the wild-type oligonucleotide reduces GATA binding by over 90%, while incubation with the mutant GATA competitor oligo has a limited effect on GATA-1, -2 and -3 binding to DNA. These results are provided for demonstration purposes only.
ab207205 has not yet been referenced specifically in any publications.