Anti-CENPE antibody [1H12] (ab5093)
- Product nameAnti-CENPE antibody [1H12]See all CENPE primary antibodies ...
- DescriptionMouse monoclonal [1H12] to CENPE
- Tested applicationsICC/IF, IP, ICC, WB, Flow Cyt more details
- Species reactivityReacts with: Human
Recombinant full length protein (Human).
- Positive controlAny human cell line should be suitable as a positive control. Kinetochore staining only visible in mitosis.
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.1% Sodium Azide
- Concentration information loading...
- Purification notesPurified from tissue culture supernatant via ion exchange chromatography (>95% total IgG).
- Clonality Monoclonal
- Clone number1H12
- Light chain typekappa
- Research Areas
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.
|ICC/IF||ICC/IF: Use at an assay dependent dilution.|
|IP||IP: Use at µg/mg of lysate. Use at a concentration of 3 µg/ml per 300 µg of lysate.|
|ICC||ICC: Use at an assay dependent dilution.|
|WB||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||Flow Cyt: Use 1µg for 106 cells.|
- RelevanceCENPE 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 localizationAssociates with kinetochores during congression, relocates to the spindle midzone at anaphase, and is quantitatively discarded at the end of the cell division.
- CENP E antibodyCentromere associated protein E antibodyCentromere autoantigen E (312kD) antibody
- Centromere autoantigen E antibodyCentromere protein E 312kDa antibodyCentromere protein E antibodyCentromeric protein E antibodyKIF10 antibodyKinesin family member 10 antibodyKinesin related protein antibodyKinesin related protein CENPE antibodyPPP1R61 antibodyProtein phosphatase 1, regulatory subunit 61 antibody
Anti-CENPE antibody [1H12] 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.
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.
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (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 for Anti-CENPE antibody [1H12] (ab5093)
This product has been referenced in:
- Hu CK et al. Midbody assembly and its regulation during cytokinesis. Mol Biol Cell 23:1024-34 (2012). ICC/IF . Read more (PubMed: 22278743) »
- Serio G et al. Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores. Proc Natl Acad Sci U S A 108:17337-42 (2011). IHC (PFA fixed) ; Human . Read more (PubMed: 21987812) »