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Immunology Adaptive Immunity T Cells Non-CD

Anti-NFAT1抗体[25A10.D6.D2] (ab2722)

  • Datasheet
  • SDS
Reviews (7)Q&A (16)References (50)

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Western blot - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Flow Cytometry - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)

Key features and details

  • Mouse monoclonal [25A10.D6.D2] to NFAT1
  • Suitable for: Flow Cyt, WB, IHC-P, ICC/IF
  • Reacts with: Human
  • Isotype: IgG1

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概述

  • 产品名称

    Anti-NFAT1抗体[25A10.D6.D2]
    参阅全部 NFAT1 一抗
  • 描述

    小鼠单克隆抗体[25A10.D6.D2] to NFAT1
  • 宿主

    Mouse
  • 特异性

    Ab2722 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.
  • 经测试应用

    适用于: Flow Cyt, WB, IHC-P, ICC/IFmore details
  • 种属反应性

    与反应: Human
  • 免疫原

    Synthetic peptide corresponding to Mouse NFAT1 aa 50-150.
    Database link: Q60591

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI
  • 阳性对照

    • 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.")
  • 常规说明

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • 存储溶液

    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • 纯度

    Protein G purified
  • 克隆

    单克隆
  • 克隆编号

    25A10.D6.D2
  • 同种型

    IgG1
  • 研究领域

    • Immunology
    • Adaptive Immunity
    • T Cells
    • Non-CD
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFATS
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Other factors
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • NFATs
    • Cardiovascular
    • Heart
    • Hypertrophy
    • Transcription factors
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Types of disease
    • Cancer

相关产品

  • ChIP Related Products

    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab2722于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
Flow Cyt
Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

WB (4)
Use a concentration of 1 µg/ml. Detects a band of approximately 120, 140 kDa (predicted molecular weight: 115 kDa).
IHC-P (1)
1/50 - 1/200.
ICC/IF
1/100 - 1/1000.
说明
Flow Cyt
Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

WB
Use a concentration of 1 µg/ml. Detects a band of approximately 120, 140 kDa (predicted molecular weight: 115 kDa).
IHC-P
1/50 - 1/200.
ICC/IF
1/100 - 1/1000.

靶标

  • 功能

    Plays 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.
  • 组织特异性

    Expressed in thymus, spleen, heart, testis, brain, placenta, muscle and pancreas.
  • 序列相似性

    Contains 1 RHD (Rel-like) domain.
  • 结构域

    Rel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors.
  • 翻译后修饰

    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.
  • 细胞定位

    Cytoplasm. 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.
  • Target information above from: UniProt accession Q13469 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 数据库链接

    • Entrez Gene: 4773 Human
    • Omim: 600490 Human
    • SwissProt: Q13469 Human
    • Unigene: 713650 Human
    • 别名

      • 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

    图片

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      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.

    • Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)

      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.

    • Western blot - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Western blot - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Anti-NFAT1 antibody [25A10.D6.D2] (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.
    • Flow Cytometry - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Flow Cytometry - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      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 - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)

      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 - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)

      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 - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)

      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [25A10.D6.D2] (ab2722)
      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.

    实验方案

    • Flow cytometry protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    数据表及文件

    • SDS download

    • Datasheet download

      Download

    文献 (50)

    发表研究结果有使用 ab2722?请让我们知道,以便我们可以引用本数据表中的参考文章。

    ab2722 被引用在 50 文献中.

    • Lao M  et al. Regulator of calcineurin 1 gene isoform 4 in pancreatic ductal adenocarcinoma regulates the progression of tumor cells. Oncogene 40:3136-3151 (2021). PubMed: 33824473
    • Cui C  et al. Propofol maintains Th17/Treg cell balance and reduces inflammation in rats with traumatic brain injury via the miR-145-3p/NFATc2/NF-?B axis. Int J Mol Med 48:N/A (2021). PubMed: 34036377
    • Vallejo-Gracia A  et al. FOXO1 promotes HIV latency by suppressing ER stress in T cells. Nat Microbiol 5:1144-1157 (2020). PubMed: 32541947
    • Wang H  et al. NF-?B-Interacting Long Noncoding RNA Regulates HIV-1 Replication and Latency by Repressing NF-?B Signaling. J Virol 94:N/A (2020). PubMed: 32581100
    • Chakraborty S  et al. A Stronger Transcription Regulatory Circuit of HIV-1C Drives the Rapid Establishment of Latency with Implications for the Direct Involvement of Tat. J Virol 94:N/A (2020). PubMed: 32669338
    View all Publications for this product

    客户评价及客户问答

    Show All 评价 Q&A
    提交评价 提交问题

    1-10 of 23 Abreviews or Q&A

    Western blot abreview for Anti-NFAT1 antibody [25A10.D6.D2]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (Heart)
    Gel Running Conditions
    Reduced Denaturing (4-12%)
    Loading amount
    30 µg
    Specification
    Heart
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    提交于 Aug 18 2020

    Immunohistochemistry (Frozen sections) abreview for Anti-NFAT1 antibody [25A10.D6.D2]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Heart)
    Permeabilization
    Yes - Tween-20
    Specification
    Heart
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    提交于 Aug 11 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-NFAT1 antibody [25A10.D6.D2] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (PDAC)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate Ph:6.0
    Permeabilization
    No
    Specification
    PDAC
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    提交于 Jul 12 2019

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

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    ChIP
    Sample
    Mouse Cell lysate - nuclear (CD4 T cells)
    Negative control
    NFAT1 ChIPseq - untreated cells. Gal4 ChIPseq - IgG control, cells treated for one hour with PMA plus ionomycin
    Specification
    CD4 T cells
    Detection step
    Other
    Type
    Native ChIP (N-ChIP)
    Positive control
    NFAT1 ChIPseq - cells treated for one hour with PMA plus ionomycin
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Dr. Giuliana Mognol

    Verified customer

    提交于 Jun 08 2017

    Western blot abreview for Anti-NFAT1 antibody [25A10.D6.D2]

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MR. Marco Craveiro

    Verified customer

    提交于 Jul 23 2013

    Question

    Would this antibody work for ChIP? Do you recommend other NFAT abs for ChIP?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 26 2012

    Answer

    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 untested application or species without financial risk.

    Simply follow these easy steps below to take part in our AbTrial Program:

    1. Reply to this e-mail to let me know that you would like to proceed and test this product.

    2. I will email you an inactive personal discount code for the value of the product.

    3. Purchase and test the product at regular price.

    4. https://www.abcam.com/abreviews with your results. Note your AbTrial discount code in the additional notes section.

    5. Once the Abreview is submitted, the discount code will become active.

    6. Apply your discount code on your next order to receive that value off.

    More information including the terms and conditions about this program may be found at: https://www.abcam.com/AbTrial

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    Abcam Scientific Support

    回复于 Nov 26 2012

    Question

    vielen Dank für Ihre Kulanz!

    Der abgelaufene Discount Code lautet xxxxx.

    Der Abreview ist für den Anti- NFAT1 (ab2722), Anonymous Abreview submitted on 11 April 2012.

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    Abcam community

    Verified customer

    Asked on Nov 26 2012

    Answer

    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.

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    Abcam Scientific Support

    回复于 Nov 26 2012

    Question

    Using spleen tissue but the band is smeary unlike the clear band shown in the image on the datasheet. How was this image made? How was the spleen lysate prepared?

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    Abcam community

    Verified customer

    Asked on Aug 03 2012

    Answer

    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 I would be happy to send a replacement antibody or issue a credit or refund.

    Please let me know if there is anything else that we can do for you. I look forward to hearing from you and resolving this as soon as possible.

    Have a nice day.

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    Abcam Scientific Support

    回复于 Aug 03 2012

    Question

    I have performed the western blotting with your antibody ab2722 as follows:

    1) I did not check the membran by Ponceau staining

    2) I did not check the gel by coomassie staining, but the protein ladder that I used was, especially within the high molecular band range, not blotted well compared to the low molecular bands

    3) Loading buffer (NuPAGEÒ LDS sample buffer) and all further reagent are from Invitrogen, 6 µg of protein were loaded per lane

    4) Transfer: 20 V ( this is the maximum for the used Xcell II Blot Modul (Invitrogen) for 4,5 h

    5) Transfer buffer: NuPAGEÒ transfer buffer from Invitrogen + 0.1% SDS + 5% Methanol

    6) PVDF membrane (pre-activated in methanol)

    7) Xcell II Blot Modul (Invitrogen), wet blotting



    Usually, for proteins smaller than 85 kDa, I perform the blotting just for 1,5 hours and it works pretty well. Furthermore, all of the ladder bands are visible quite good in this case.



    It would be very helpfull if you could provide the exact protocol from the testing of this antibody as indicated in the antibody datasheet!



    With kind regards,

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    Abcam community

    Verified customer

    Asked on Jul 03 2012

    Answer

    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. I am sorry to repeat myself, however the transfer conditions are indeed dependent on the equipment used.

    The laboratory also recommend to transfer over night and to use 0.01% SDS in the transfer buffer.

    Please let me know how you are proceeding. I would be pleased to discuss the optimization further with you.

    If you really want to know transfer conditions, you still could contact authors of the publications or Abreviews we do have listed on our website for this antibody.

    I am looking forward to hear back from you. Please do not hesitate to contact me also again, should you have any other questions or concerns.

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    Abcam Scientific Support

    回复于 Jul 03 2012

    Question

    I have performed the western blotting with your antibody ab2722 as follows:


    1) I did not check the membran by Ponceau staining

    2) I did not check the gel by coomassie staining, but the protein ladder that I used was, especially within the high molecular band range, not blotted well compared to the low molecular bands

    3) Loading buffer (NuPAGEÒ LDS sample buffer) and all further reagent are from Invitrogen, 6 µg of protein were loaded per lane

    4) Transfer: 20 V ( this is the maximum for the used Xcell II Blot Modul (Invitrogen) for 4,5 h

    5) Transfer buffer: NuPAGEÒ transfer buffer from Invitrogen + 0.1% SDS + 5% Methanol

    6) PVDF membrane (pre-activated in methanol)

    7) Xcell II Blot Modul (Invitrogen), wet blotting

    Usually, for proteins smaller than 85 kDa, I perform the blotting just for 1,5 hours and it works pretty well. Furthermore, all of the ladder bands are visible quite good in this case.


    It would be very helpfull if you could provide the exact protocol from the testing of this antibody as indicated in the antibody datasheet!



    With kind regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jun 29 2012

    Answer

    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 blotting depends on the material you are using and is not dependent on the antibody. The western blot transfer should be optimized in your lab according to your conditions and results, as they might differ from laboratory to laboratory.

    I can also strongly recommend to perform ponceau red stainings. This is a very fast and reliable method to check the transfer efficiency.

    For high molecular weight proteins, I can recommend the following:

    - I would highly suggest to optimize the transfer conditions by increasing the transfer time to 18 or 24 hours (in some examples, for big proteins, the transfer is done for 48 hours)

    - A transfer at 20V for a long period of time is fine but it has been shown that a "boost" at 70-80V for 1 hour at the end of the transfer can be of significant improvement.

    -I do not know the exact composition of your buffer, however you can use up to 0.05%SDS in the transfer buffer and reduce the proportion of methanol to 10%. The reason is that methanol washes out the SDS from the protein.

    I hope this information is helpful. Please let me know if you have any question.

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    Abcam Scientific Support

    回复于 Jun 29 2012

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