GATA (GATA1, GATA2, GATA3) Transcription Factor Assay Kit (Colorimetric) (ab207205)

Overview

  • Product name
    GATA (GATA1, GATA2, GATA3) Transcription Factor Assay Kit (Colorimetric)
  • Detection method
    Colorimetric
  • Sample type
    Nuclear Extracts
  • Assay type
    Semi-quantitative
  • Sensitivity
    < 600 ng/well
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Human
  • Product overview

    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.

  • Notes

    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.

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Please 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
  • Function
    Transcriptional 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).
  • Tissue specificity
    Erythrocytes.
  • Involvement in disease
    X-linked dyserythropoietic anemia and thrombocytopenia
    Thrombocytopenia with beta-thalassemia, X-linked
    Anemia without thrombocytopenia, X-linked
  • Sequence similarities
    Contains 2 GATA-type zinc fingers.
  • Domain
    The 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.
  • Post-translational
    modifications
    Highly 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.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Alternative names
    • Eryf1
    • Erythroid transcription factor
    • GATA 2
    • GATA 3
    • GATA-1
    • GATA-binding factor 1
    • GATA1
    • GATA1_HUMAN
    • GF-1
    • NF-E1 DNA-binding protein
    see all
  • Database links

Images

  • 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.

Protocols

References

ab207205 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

There are currently no Customer reviews or Questions for ab207205.
Please use the links above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up