Overview

  • Product name

    NFATc1 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: Mouse, Rat, Human
  • Product overview

    NFATc1 Transcription Factor Assay Kit (Colorimetric) (ab207215) is a high throughput assay to quantify NFATc1 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 NFATc1 consensus binding site (5’ –AGGAAA– 3’) has been immobilized onto a 96-well plate. Active NFATc1 present in the nuclear extract specifically binds to the oligonucleotide. NFATc1 is detected by a primary antibody that recognizes an epitope of NFATc1 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 human and mouse NFATc1.


    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 – 10 µg nuclear extract/well.

  • Notes

    NFAT proteins are transcription factors that were first identified as inducers of the immune response. As demonstrated later, these proteins also play varied roles in cell differentiation and adaptation for vascular endothelial cells or skeletal muscle cells. There are four NFAT family members (NFAT1-4, NFAT2 is also called NFATc or NFATc1) from which numerous isoforms are generated by alternative splicing1. NFAT mRNAs are found in peripheral blood lymphocytes, spleen (NFAT1 and 2) and thymus (NFAT4). NFATs modulate the expression of numerous cytokines such as IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, GM-CSF, IFNa, IFNg and CD40L. In resting cells, NFATc1 is confined to the cytoplasm, where it is maintained in a phosphorylated state by the action of constitutive kinases. Upon stimulation, NFATc1 is dephosphorylated by calcineurin, a Ca2+-dependent phosphatase, and migrates to the nucleus. NFATc1 dephosphorylation is stimulated by Ca2+-coupled membrane receptors, such as T cell and B cell receptors, and the CD40, FceRI, CD16 and G protein-associated receptors (thrombin or H1 histamine receptors). NFATc1 can also be activated by calcium ionophores. Cyclosporin A and FK506 immunosuppressor drugs inhibit calcineurin activity on NFAT. Receptors not associated with calcium movement are not expected to stimulate NFAT. When calcium levels drop, calcineurin becomes inactive, and NFATc1 is rephosphorylated by kinases and exported back into the cytoplasm.

    NFAT phosphoproteins share two conserved domains:  a DNA-binding domain (DBD) displaying limited similarity to the Rel protein family DBD, and modulating interactions with AP-1 dimers; and a NFAT homology region (NHR), upstream of the DBD that regulates translocation and DNA-binding activity. The regulatory domain is dephosphorylated by the calcium- and calmodulin-dependent phosphatase calcineurin, which controls NFAT nuclear translocation. Transactivation domains can be found at the N- and C-terminal ends of the NFAT proteins. NFATs bind to the DNA consensus motif 5´-T/AGGAAA-3´ as monomers. NFATs can cooperatively interact with AP-1 and GATA proteins for DNA binding. NFAT can also bind to certain kB-like sites. Vitamin D3 receptor heterodimers (RXR:VDR) can abrogate NFAT modulation of IL-2 by binding to a site which overlaps the NFAT distal site. Sites in IL-2 and GM-CSF promoters can accommodate both NFAT and the Ets-family member, Elf-1.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well NFAT assay plate 1 unit 5 units
    Anti-mouse HRP-conjugated IgG (0.4 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    Jurkat nuclear extract (2.5 μg/μL) 1 x 40µl 1 x 200µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    Mutated oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
    NFATc1 antibody (0.2 μg/μL) 1 x 22µl 1 x 110µl
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide (10 pmol/μL) 1 x 100µl 1 x 500µl
  • Research areas

  • Relevance

    NFATc1 is a component of the nuclear factor of activated T cells DNA-binding transcription complex. This complex consists of at least two components: a preexisting cytosolic component that translocates to the nucleus upon T cell receptor (TCR) stimulation, and an inducible nuclear component. Proteins belonging to this family of transcription factors play a central role in inducible gene transcription during immune response. This protein is an inducible nuclear component. It functions as a major molecular target for the immunosuppressive drugs such as cyclosporin A. Different isoforms may regulate inducible expression of different cytokine genes.
  • Cellular localization

    Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation.
  • Alternative names

    • NAFATC
    • NFAT transcription complex cytosolic component
    • NFAT2
    • NFATC 1
    • NFATC1
    • Nuclear factor of activated T cells cytoplasmic 1
    see all
  • Database links

Images

  • Different amounts of nuclear extract from untreated (light grey) and PHA treated (dark grey) Jurkat cells were tested for NFATc1 activation. This data is provided for demonstration only.

Protocols

References

This product has been referenced in:

  • Wang Q  et al. Raddeanin A suppresses breast cancer-associated osteolysis through inhibiting osteoclasts and breast cancer cells. Cell Death Dis 9:376 (2018). Read more (PubMed: 29515110) »
See 1 Publication for this product

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