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H2AFX 抗体 (pSer139) (Alexa Fluor 647)

H2AFX 适用: 人, 小鼠 BI, ICS 宿主: 小鼠 Monoclonal N1-431 Alexa Fluor 647
产品编号 ABIN1177064
发货至: 中国
  • 抗原 See all H2AFX 抗体
    H2AFX (H2A Histone Family, Member X (H2AFX))
    抗原表位
    • 44
    • 27
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    pSer139
    适用
    • 184
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    人, 小鼠
    宿主
    • 163
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    小鼠
    克隆类型
    • 128
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    单克隆
    标记
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    This H2AFX antibody is conjugated to Alexa Fluor 647
    应用范围
    • 147
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    BioImaging (BI), Intracellular Staining (ICS)
    品牌
    BD Pharmingen™
    纯化方法
    The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
    免疫原
    Phosphorylated Human H2AX Peptide
    克隆位点
    N1-431
    亚型
    IgG1 kappa
    Top Product
    Discover our top product H2AFX Primary Antibody
  • 样本量
    5 μL
    实验流程
    1. Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and culture overnight to 48 hours.
    2. Remove the culture medium from the wells, and wash (one to two times) with 100 myl of 1x PBS.
    3. Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).
    4. Remove the fixative from the wells, and wash the wells (one to two times) with 100 myl of 1x PBS.
    5. Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):
    a. Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
    b. Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
    c. Add 100 µl of 1x Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT. Continue to use 1x Perm/Wash buffer for all subsequent wash and dilutions steps.
    6. Remove the permeabilization buffer from the wells, and wash one to two times with 100 myl of appropriate buffer (either 1x PBS or 1x Perm/Wash buffer, see step 5.c.).
    7. Optional blocking step: Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.
    8. Dilute the antibody to its optimal working concentration in appropriate dilution buffer. Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration. If using a Bioimaging Certified antibody conjugate, dilute it 1:10.
    9. Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT. Incubate in the dark if using fluorescently labeled antibodies.
    10. Remove the antibody, and wash the wells three times with 100 myl of wash buffer. An optional detergent wash (100 myl of 0.05% Tween in 1x PBS) can be included prior to the regular wash steps.
    11. If the antibody being used is fluorescently labeled, then move to step 12. Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.
    12. After the final wash, counter-stain the nuclei by adding 100 ml of a 2 mg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1x PBS to each well at least 15 minutes before imaging.
    13. View and analyze the cells on an appropriate imaging instrument.
    限制
    仅限研究用
  • 状态
    Liquid
    缓冲液
    Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09 % sodium azide.
    储存液
    Sodium azide
    注意事项
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    储存条件
    4 °C
    储存方法
    The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
  • Kuo, Yang: "Gamma-H2AX - a novel biomarker for DNA double-strand breaks." in: In vivo (Athens, Greece), Vol. 22, Issue 3, pp. 305-9, (2008) (PubMed).

    Fernandez-Capetillo, Lee, Nussenzweig, Nussenzweig: "H2AX: the histone guardian of the genome." in: DNA repair, Vol. 3, Issue 8-9, pp. 959-67, (2004) (PubMed).

    Burma, Chen, Murphy, Kurimasa, Chen: "ATM phosphorylates histone H2AX in response to DNA double-strand breaks." in: The Journal of biological chemistry, Vol. 276, Issue 45, pp. 42462-7, (2001) (PubMed).

    Rogakou, Nieves-Neira, Boon, Pommier, Bonner: "Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139." in: The Journal of biological chemistry, Vol. 275, Issue 13, pp. 9390-5, (2000) (PubMed).

    Rogakou, Pilch, Orr, Ivanova, Bonner: "DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139." in: The Journal of biological chemistry, Vol. 273, Issue 10, pp. 5858-68, (1998) (PubMed).

  • 抗原
    H2AFX (H2A Histone Family, Member X (H2AFX))
    别名
    H2AX (H2AFX 产品)
    别名
    H2A.X antibody, H2A/X antibody, H2AX antibody, AW228881 antibody, H2ax antibody, Hist5-2ax antibody, gammaH2ax antibody, zgc:56329 antibody, h2a.x antibody, h2a/x antibody, h2ax antibody, RGD1566119 antibody, h2a antibody, h2afx antibody, H2A histone family member X antibody, H2A histone family, member X antibody, H2A histone family member X L homeolog antibody, histone cluster 1, H2ah antibody, histone cluster 2, H2ab S homeolog antibody, histone H2AX antibody, H2AFX antibody, H2afx antibody, h2afx antibody, h2afx.L antibody, HIST1H2AH antibody, hist2h2ab.S antibody, LOC100522201 antibody, LOC100720536 antibody
    背景
    Histones are highly basic proteins that complex with DNA to form chromatin. The H2AX histone (~15 kDa calculated molecular weight) is a member of the H2A histone family whose members are components of nucleosomal histone octamers. Double-stranded breaks in DNA caused by replication errors, apoptosis, or other physiological processes (including, immunoglobulin and TCR gene recombinations) and DNA damage caused by ionizing radiation, UV light, or cytotoxic agents lead to phosphorylation of H2AX on serine 139. H2AX (pS139) is also referred to as H2AX (pS140) when the N-terminal methionine that is normally excised during posttranslational processing is included in amino acid sequence numbering. Kinases such as ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) phosphorylate H2AX to induce its function. Phosphorylated H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to the DNA-damaged sites. In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability and thus helps prevent oncogenic transformations. Immunofluorescent staining and bioimaging analysis of cultured cells can be used to readily identify H2AX (pS139)-containing foci. As such, H2AX (pS139) immunofluorescence localization serves as a biomarker for nuclear sites of DNA damage (e.g., double-stranded DNA breaks) in affected cells. Immunofluorescent staining of human cell line. HeLa cells (ATCC CCL-2) were seeded in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) at ~10,000 cells per well. After overnight culture, the cells were exposed to 2400 Joules UV irradiation (right image) or untreated (left image) and then allowed to recover for 30-60 minutes at 37°C. The cells were fixed, permeabilized with cold methanol, and stained with Alexa Fluor® 647 Mouse anti-H2AX (pS139) (pseudo colored magenta) according to the Recommended Assay Procedure. Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The images were captured on a BD Pathway™ 435 high-content Bioimager system using a 20X objective and merged using BD AttoVision™ software. This antibody also worked with the Saponin and the Triton™ X-100 Perm/Wash protocols.
    Synonyms: H2A.X, H2A/X, H2AFX, HIST5-2AX, gamma-H2AX, gamma-H2AX, H2AX (pS140)
    途径
    Telomere Maintenance, DNA Damage Repair, Positive Regulation of Response to DNA Damage Stimulus
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