Anti-CENPE antibody [1H12]

• Flow Cyt

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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.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (AB5093)

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.

This image is courtesy of Scott Slattery and Mike Mancini

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CENPE antibody [1H12] (AB5093)

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.

This image is courtesy of an Abreview submitted by Dr Beth Sullivan

• ICC/IF

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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.

• WB

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Western blot - Anti-CENPE antibody [1H12] (AB5093)

All lanes:

Western blot - Anti-CENPE antibody [1H12] (ab5093) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 316 kDa

Observed band size: 270 kDa

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Exposure time: 20min