Anti-CENPE抗体[1H12] (ab5093)

Reviews (4)Q&A (2)References (31)
Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
  • Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
  • Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
  • Western blot - Anti-CENPE antibody [1H12] (ab5093)
  • Flow Cytometry - Anti-CENPE antibody [1H12] (ab5093)

Key features and details

  • Mouse monoclonal [1H12] to CENPE
  • Suitable for: ICC/IF, WB, Flow Cyt
  • Reacts with: Human
  • Isotype: IgG1

概述

  • 产品名称

    Anti-CENPE抗体[1H12]
    参阅全部 CENPE 一抗

  • 描述

    小鼠单克隆抗体[1H12] to CENPE

  • 宿主

    Mouse

  • 经测试应用

    适用于: ICC/IF, WB, Flow Cytmore details

  • 种属反应性

    与反应: Human

  • 免疫原

    Recombinant full length protein (Human).

  • 阳性对照

    • Any human cell line should be suitable as a positive control. Kinetochore staining only visible in mitosis.

  • 常规说明

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    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, as well as customer reviews and Q&As.

性能

应用

Our Abpromise guarantee covers the use of ab5093 in the following tested applications.

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

应用 Ab评论 说明
ICC/IF Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 1 µg/ml. Predicted molecular weight: 312 kDa. Only suitable for WB if IP is performed first.
Flow Cyt Use 1µg for 106 cells.

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

靶标

  • 相关性

    CENPE is a 250-300 kDa human centromere-associated kinesin-like motor protein that accumulates in G2 phase. In contrast to other centromere proteins, CENPE is not detected at centromeres during interphase, and first appears at the centromere region of chromosomes during prometaphase. CENPE function is required for the transition from metaphase to anaphase. CENPE is probably one of the motors responsible for mammalian chromosome movement and/or spindle elongation.

  • 细胞定位

    Associates with kinetochores during congression, relocates to the spindle midzone at anaphase, and is quantitatively discarded at the end of the cell division.

  • 数据库链接

    • 别名

      • CENP E antibody
      • Centromere associated protein E antibody
      • Centromere autoantigen E (312kD) antibody
      • Centromere autoantigen E antibody
      • Centromere protein E 312kDa antibody
      • Centromere protein E antibody
      • KIF10 antibody
      • Kinesin family member 10 antibody
      • Kinesin related protein antibody
      • Kinesin related protein CENPE antibody
      • PPP1R61 antibody
      • Protein phosphatase 1, regulatory subunit 61 antibody
      see all

    图片

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)

      Kinetochores specific staining of HCT116 cells arrested in G2/M phase by nocodazole treatment. Methanol fixed cells were stained using mouse monoclonal [1H12] antibody to CENP-E ab5093 (green) and DAPI (blue).

      This image was kindly supplied as part of the review submitted by Salvador Rodrigez-Nieto.

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)This image is courtesy of an Abreview submitted by Dr Beth Sullivan
      ab5093 at 1/500 staining human fibrosarcoma (HT1080) cells by ICC/IF. The cells were treated with 0.1-0.2ug/mL colcemid for 45-60 minutes, then swollen in hypotonic buffer for 8 minutes and centrifuged onto glass slides. Cells were blocked in 1X PBS + 1% BSA + 0.5% Triton X-100 (blocking buffer) for 30 minutes at room temperature. The antibodies were diluted 1/300-1/500 in blocking buffer and incubated overnight at 4 degrees C. ab5093 was detected with Alexa Fluor 488-donkey anti-mouse for 1-2 hours at room temperature.

      See Abreview

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (ab5093)This image is courtesy of Scott Slattery and Mike Mancini
      HeLa cells were stained with ab5093, anti-CENPE (in green) in panel one, and with ab5093 and SH-CREST (red), which stains the centromeres, in panel 2. Fix cells for 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 minutes with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM. Permeablize 30 minutes with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody overnight at 4oC diluted 1/250 in 5% milk in TBST. Secondary antibody was incubated for 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 minutes with ammonium chloride in PEM. Counterstain with DAPI in TBST. Mount with ProLong Gold antifade reagent from Invitrogen. Notes: Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2.
    • Western blot - Anti-CENPE antibody [1H12] (ab5093)
      Western blot - Anti-CENPE antibody [1H12] (ab5093)
      All lanes : Anti-CENPE antibody [1H12] (ab5093) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
      Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : 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: 312 kDa
      Observed band size: 270 kDa
      why is the actual band size different from the predicted?


      Exposure time: 20 minutes
    • Flow Cytometry - Anti-CENPE antibody [1H12] (ab5093)
      Flow Cytometry - Anti-CENPE antibody [1H12] (ab5093)
      Overlay histogram showing HeLA cells stained with ab5093 (red line). The cells were fixed with 100% 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 (ab5093, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

    文献 (31)

    发表研究结果有使用 ab5093？请让我们知道，以便我们可以引用本数据表中的参考文章。

    ab5093 被引用在 31 文献中.

    • Waseem NH  et al. Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma. PLoS Genet 16:e1008721 (2020). PubMed: 32339198
    • Tan Z  et al. Environmental stresses induce karyotypic instability in colorectal cancer cells. Mol Biol Cell 30:42-55 (2019). PubMed: 30379607
    • Wu M  et al. LUBAC controls chromosome alignment by targeting CENP-E to attached kinetochores. Nat Commun 10:273 (2019). PubMed: 30655516
    • Sikirzhytski V  et al. Microtubules assemble near most kinetochores during early prometaphase in human cells. J Cell Biol 217:2647-2659 (2018). PubMed: 29907657
    • Gurden MD  et al. Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis. Oncotarget 9:19525-19542 (2018). PubMed: 29731963
    View all Publications for this product

    客户评价及客户问答

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    1-6 of 6 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPE antibody [1H12]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Nocodazole (100ng/mL) treated NHDF)
    Permeabilization
    Yes - 0.5% Triton
    Specification
    Nocodazole (100ng/mL) treated NHDF
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 27°C
    Fixative
    Extraction with calcium buffer (100 mM pipes, 1 mM MgCl2, 1 mM CaCl2, and 0.5% Triton X-100 at pH 6.8) at room temperature for 3–5 min, then fixed in 4% paraformaldehyde
    Read More

    Dana Mitchell

    Verified customer

    提交于 Nov 02 2020

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPE antibody [1H12]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Methanol
    Permeabilization
    No
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 27°C
    Read More

    Abcam user community

    Verified customer

    提交于 Dec 16 2008

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPE antibody [1H12]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Methanol
    Permeabilization
    No
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3.0% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    提交于 Nov 18 2008

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPE antibody [1H12]

     Good
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (fibrosarcoma (HT1080))
    Specification
    fibrosarcoma (HT1080)
    Fixative
    Methanol
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 1%
    Read More

    Dr. Beth Sullivan

    Verified customer

    提交于 Jan 17 2007

    Question

    BATCH NUMBER 135498 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE HeLa, 40~50 ug, whole cell PRIMARY ANTIBODY 1:500, 1:250, (ON at 4C) in 5%NFDM Wash in TBS-T 3X 5 minutes DETECTION METHOD ECL from Pierce POSITIVE AND NEGATIVE CONTROLS USED HeLa (+) ANTIBODY STORAGE CONDITIONS aliquotted and stored at -20C SAMPLE PREPARATION Boiled in SDS AMOUNT OF PROTEIN LOADED 40~50 ug ELECTROPHORESIS/GEL CONDITIONS 6% reducing TRANSFER AND BLOCKING CONDITIONS semi-dry, 20V 35, 40, 45, 50 minutes (4 different times) 5% NFDM SECONDARY ANTIBODY 5% NFDM 1 hr at RT anti-mouse; 1:1,000~1:2,000 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Dilutions of primary; transfer times ADDITIONAL NOTES Secondary shown to work on another primary; some background but no major bands

    Read More
    Answer

    Thank you for your enquiry. I am sorry to hear that one of your customers has been having difficulties with this antiserum. I have had a look at your submitted technical questionnaire and I have a few comments. Your customers western blot protocol is not significantly different from one I would recommend. I would like to suggest the use of 5% BSA as a blocking solution or even PBST or TBST alone to improve your chances of signal. It may be that your customer is out competing the binding of the antiserum and therefore achieving poor signal. According to our datasheet "this antibody is only suitable for WB if IP is performed first". Therefore it is highly likely that your customer is unable to detect this protein by western blot on whole cell HeLa lysates because it is beyond the detection threshold of this antiserum, as described on our datasheet. I hope this information helps. Please do not hesitate to contact me should you require further assistance.

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    Question

    What IP conditions should be used?

    Read More
    Answer

    Standard conditions such as RIPA buffer or NP40 lysis buffer (0.5 to 1% NP40, PBS +/- glycerol).

    Read More

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