Anti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade (ab2722)

Overview

  • Product nameAnti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade
    See all NFAT1 primary antibodies
  • Description
    Mouse monoclonal [25A10.D6.D2] to NFAT1 - ChIP Grade
  • SpecificityAb2722 detects nuclear factor of activated T-cells (NFAT) from mouse, rat and human tissues (endogenously expressed). This antibody does not cross react with NFAT2 (NFATc, NFATc1). This antibody detects both forms NFAT1 - a ~140 kDa protein representing phosphorylated NFAT1 in resting immune cells, and a ~120 kDa protein in stimulated cells that represents fully-dephosphorylated NFAT1.
  • Tested applicationsSuitable for: IHC-P, Flow Cyt, Gel supershift assays, ICC, IP, WB, IHC-Fr, ICC/IF, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Mouse NFAT1 aa 51-69.
    Sequence:

    AISSPSGLAYPDDVLDYGL


    (Peptide available as ab4905)

  • Positive control
    • WB: resting immune cells and ionomycin stimulated immune cells (see Shaw et al reference: "1uM for T cells and B cells, 10uM for macrophages,and 0.3uM for mast cells. Treatment was for 20 min.")

Properties

Applications

Our Abpromise guarantee covers the use of ab2722 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P 1/125. PubMed: 19828810
Flow Cyt Use at an assay dependent concentration.
Gel supershift assays Use at an assay dependent concentration. PubMed: 12065418
ICC 1/100 - 1/1000.

Fix with 3% paraformaldehyde at 37°C or with ice-cold methanol (see Shaw et al reference (clone refered to as 67.1).

IP Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 120, 140 kDa (predicted molecular weight: 115 kDa).Can be blocked with NFAT1 peptide (ab4905).
IHC-Fr 1/50 - 1/200.
ICC/IF 1/100 - 1/1000.
ChIP Use at an assay dependent concentration. PubMed: 22989874

Target

  • FunctionPlays a role in the inducible expression of cytokine genes in T-cells, especially in the induction of the IL-2, IL-3, IL-4, TNF-alpha or GM-CSF.
  • Tissue specificityExpressed in thymus, spleen, heart, testis, brain, placenta, muscle and pancreas.
  • Sequence similaritiesContains 1 RHD (Rel-like) domain.
  • DomainRel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors.
  • Post-translational
    modifications
    In resting cells, phosphorylated by NFATC-kinase on at least 18 sites in the 99-363 region. Upon cell stimulation, all these sites except Ser-243 are dephosphorylated by calcineurin. Dephosphorylation induces a conformational change that simultaneously exposes an NLS and masks an NES, which results in nuclear localization. Simultaneously, Ser-53 or Ser-56 is phosphorylated; which is required for full transcriptional activity.
  • Cellular localizationCytoplasm. Nucleus. Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation. Rapid nuclear exit of NFATC is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. The subcellular localization of NFATC plays a key role in the regulation of gene transcription.
  • Information by UniProt
  • Database links
  • Alternative names
    • AI607462 antibody
    • cytoplasmic 2 antibody
    • KIAA0611 antibody
    • NF ATc2 antibody
    • NF ATp antibody
    • NF-ATc2 antibody
    • NF-ATp antibody
    • NFAC2_HUMAN antibody
    • NFAT 1 antibody
    • NFAT pre existing subunit antibody
    • NFAT pre-existing subunit antibody
    • NFAT transcription complex, preexisting component antibody
    • NFAT1 antibody
    • NFAT1-D antibody
    • NFATc2 antibody
    • NFATp antibody
    • Nuclear factor of activated T cells cytoplasmic 2 antibody
    • Nuclear factor of activated T cells cytoplasmic calcineurin dependent 2 antibody
    • Nuclear factor of activated T cells pre-existing component antibody
    • Nuclear factor of activated T cells, preexisting component antibody
    • Nuclear factor of activated T-cells antibody
    • Preexisting nuclear factor of activated T cells 2 antibody
    • T cell transcription factor NFAT 1 antibody
    • T-cell transcription factor NFAT1 antibody
    see all

Anti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade images

  • ab2722 (4µg/ml) staining NFAT in human tonsil, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and weak cytoplasmic staining.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Immunocytochemistry/Immunofluorescence analysis of NFAT1 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were left untreated (left panel) or treated with 1uM staurosporine (right panel) for 3 hours and incubated with ab2722 (1:100) for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488 conjugated goat anti-mouse IgG secondary antibody (1:400) for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NFAT1 (green) shows staining in HeLa cells. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were inbuated without (control) or with ab2722 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NFAT1 (green) shows staining in MCF-7 cells. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were inbuated without (control) or with ab2722 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NFAT1 (green) shows staining in U251 Cells. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were inbuated without (control) or with ab2722 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Anti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade (ab2722) at 1 µg/ml + Human spleen tissue lysate - total protein (ab29699) at 10 µg

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution ( )
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 115 kDa
    Observed band size : 150 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 62 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 4 minutes
    NFAT1 contains an exstensive number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
  • Overlay histogram showing Jurkat cells stained with ab2722 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2722, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human colon carcinoma tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing NFATc2 ab2722 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human spleen tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing NFATc2 ab2722 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human tonsil tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing NFATc2 ab2722 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

References for Anti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade (ab2722)

This product has been referenced in:
  • Makowski SL  et al. A protease-independent function for SPPL3 in NFAT activation. Mol Cell Biol 35:451-67 (2015). Read more (PubMed: 25384971) »
  • Lodhi N  et al. Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark. Nucleic Acids Res 42:7028-38 (2014). ChIP ; Human . Read more (PubMed: 24861619) »

See all 15 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Human Purified protein (Human primary CD4 T cells)
Specification Human primary CD4 T cells
Treatment TCR activated using anti-CD3/CD28 for 3,10 and 30min
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Mr. Marco Craveiro

Verified customer

Submitted Jul 23 2013

Thank you very much for your interest in ab92490.

To our knowledge, this product nor any of our other anti-NFAT products, have not been tested in ChIP. However by participating in our AbTrial program, you can now use our products in an unt...

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Vielen dank für die Bestätigung des Codes und des Abreviews.

Ihr neuer Code lautet: xxxxxxx.

Der Code ist über einen Wert von €410.00 und gültig bis zum 26/03/2013.

Thank you for taking the time to complete our recent phone survey about your experiences with Abcam.

I'd like to follow-up on this issue since the problem has not been resolved at this point. If the protocol suggestionswere not helpful, then ...

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Thank you for your patience.

The laboratory has got back to me - however unfortunately they do not have the protocol file for transfer. I am sorry. I hope however with the tips I gave you last time, you were able to optimize the Western blot....

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Thank you for your answers. I have written to the laboratory to see whether they have the protocol which they have had used. I will get back to you as soon as I have a reply.

I would like however to re-iterate that the efficiency of the blott...

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Thank you very much for your inquiry.

I am sorry that you are experiencing problems in Western blotting. The transfer conditions of the Western blot depend mostly on the machinery used. I would therefore recommend to check what programms of t...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (el4)
Loading amount 100000 cells
Specification el4
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

Submitted Jun 12 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Lymphocte)
Loading amount 600000 cells
Specification Lymphocte
Gel Running Conditions Reduced Denaturing (12)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Apr 11 2012

Vielen Dank für Ihre Rückmeldung.

Ich freue mich wirklich, dass Sie mit dem Ersatz so schöne Ergebnisse bekommen haben!

Den kostenlosen Primärantikörper können Sie mit einem Gutscheincode von uns erhalt...

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