Overview

  • Product name
    Anti-CENPE antibody [1H12]
    See all CENPE primary antibodies
  • Description
    Mouse monoclonal [1H12] to CENPE
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, IP, ICC, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein (Human).

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

Properties

Applications

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.

Application Abreviews Notes
ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration. Use at a concentration of 3 µg/ml per 300 µg of lysate.
ICC 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.

Target

  • Relevance
    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.
  • Cellular localization
    Associates with kinetochores during congression, relocates to the spindle midzone at anaphase, and is quantitatively discarded at the end of the cell division.
  • Database links
  • Alternative names
    • 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

Images

  • 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.

  • 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

  • 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.
  • 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
  • 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.

References

This product has been referenced in:
  • 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). Read more (PubMed: 29731963) »
  • Worrall JT  et al. Non-random Mis-segregation of Human Chromosomes. Cell Rep 23:3366-3380 (2018). Read more (PubMed: 29898405) »
See all 23 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Dec 16 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Nov 18 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
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%

Dr. Beth Sullivan

Verified customer

Submitted Jan 17 2007

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
Answer

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

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