Anti-HEC1/HEC antibody [9G3]

• ICC/IF

AbReview5801****

Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

ab3613 at 1/1000 dilution staining HeLa cells by ICC/IF. The cells were formaldehyde fixed and blocked with 5% BSA prior to incubation with the antibody for 2 hours. An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary.

This image is courtesy of an anonymous Abreview

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

Immunofluorescence analysis of HeLa cells fixed in 4% paraformaldehyde at RT for 15 min labelling Hec1 protein at kinetochore using ab3613 at a 1/1000 dilution (Green). alpha Tubulin, a cytoskeleton marker, stained by alpha Tubulin antibody used at a 1/1000 dilution (Red). DAPI was used for nuclear staining (Blue).

• Flow Cyt

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Flow Cytometry - Anti-HEC1/HEC antibody [9G3] (AB3613)

Overlay histogram showing HeLa cells stained with ab3613 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab3613, 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 IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min) /permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

Immunofluorescence analysis of HeLa cells fixed in 4% paraformaldehyde at RT for 15 min labelling Hec1 protein at kinetochore using ab3613 at a 1/500 dilution (Green). beta Tubulin, a cytoskeleton marker, stained by beta Tubulin antibody used at a 1/1000 dilution (Red). DAPI was used for nuclear staining (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling HEC1/HEC at the kinetochore with ab3613 at 1/500 (green). Cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes. The cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 4 minutes. The cells were blocked in 2.5% BSA/PBS at room temperature for 30 minutes. ab3613 was incubated at 4°C overnight. The secondary antibody was a Rabbit IgG antibody (Alexa Fluor 488), 1/2000 (keep from light), room temperature for 1hour. Washing with PBS 3 x 3 minutes. Red : alpha Tubulin 4a, a cytoskeleton marker, stained by an alpha Tubulin 4a antibody.
Blue : Hoechst 33342 staining.

Synchronized condition : Suggest to treat cells with Nocodazole (10ng/ml, 24hr).

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

HeLa cells were stained with anti-HEC1/HEC (ab2613; in green) and DAPI (blue) in panel 1, and with anti-HEC1/HEC (green) and SH-CREST (red) to stain the centromeres in panel 2. Fix the cells 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM. Permeablize 30 min. with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody incubated at 1/1000 overnight at 4oC diluted in 5% milk in TBST. Secondary antibody 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 min. on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

Immunofluorescence analysis of HeLa cells fixed in 4% paraformaldehyde at RT for 15 min labelling Hec1 protein at kinetochore using ab3613 at a 1/500 dilution (Green). DAPI was used for nuclear staining (Blue).

• WB

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Western blot - Anti-HEC1/HEC antibody [9G3] (AB3613)

7.5% gel.

Running condition : 80V, 15min; 140V, 40min.

Transfer condition : Semi-dry, 18 V, 60min (Nitrocellulose membrane).

Blocking condition : 5% non-fat milk in TBST, RT, 60min.

Primary antibody incubation : 4°C overnight.

Secondary antibody incubation : Room temperature for 1 hour.

Washing condition : 5 ml TBST, 4 x 5min.

ECL detection.

All lanes:

Western blot - Anti-HEC1/HEC antibody [9G3] (ab3613) at 1/1000 dilution

Lane 1:

293T whole cell lysate at 30 µg

Lane 2:

A431 whole cell lysate at 30 µg

Lane 3:

HepG2 whole cell lysate at 30 µg

Lane 4:

HeLa whole cell lysate at 30 µg

Lane 5:

HeLa nuclear extract at 30 µg

Secondary

All lanes:

Mouse IgG antibody (HRP) at 1/5000 dilution

Predicted band size: 74 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HEC1/HEC antibody [9G3] (AB3613)

Anti-HEC1/HEC antibody (ab3613) labels the kinetochores of mitotic cells in LLCPK1 (Sus scrofa kidney epithelial cell line) cell lines. Merge shows an overlay of DNA (stained with DAPI, red) and HEC1/HEC (green).
This image was kindly supplied as part of the review submitted by Marko Kallio.

• WB

CiteAb

Western blot - Anti-HEC1/HEC antibody [9G3] (AB3613)

HEC1/HEC western blot using anti-HEC1/HEC antibody [9G3] ab3613. Publication image and figure legend from Tan, E. P., Caro, S., et al., 2013, J Biol Chem, PubMed 23946484.

ab3613 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab3613 please see the product overview.

OGT or OGA gain of function disrupts spindle architecture. a, GFP, OGT, or OGA gain of function or OGA inhibitor Thiamet-G-treated HeLa cells were synchronized to M phase. Western blots were performed for O-GlcNAc, OGA, OGT, GFP, cyclin B, and actin. b–d, DNA (blue) and α-tubulin (green) confocal imaging of M phase gain of function OGT/OGA cells, or cells treated with Thiamet-G. Midzone width and length (mean ± S.E., replicate number (n) : nGFP = 195, nOGT = 193, nOGA = 198, nNT = 195, nTMG = 167, *, p < 0.005 between GFP/NT versus OGT/OGA or TMG) were quantified using ImageJ software as follows : first, the condensed chromatin area color was inverted in order to delineate the edge of the chromatin. Next, the chromatin was measured lengthwise, and the width was measured three times from different sections and averaged. Yellow lines are a representative measurement. e, multipolar spindles were quantified from confocal images of DNA (blue) and γ-tubulin (green) M phase synchronized gain of function OGT/OGA cells. f, DNA (blue) and γ-tubulin (green) were confocal imaged at M phase in OGT/OGA gain of function cells. Distance between each centrosome and the midzone was quantified (mean ± S.E., replicate number (n) : nControl = 93, nOGT = 64, nOGA = 76, *, p < 0.005 between Control versus OGT/OGA). g, spindle angle measurement schematic in OGT/OGA gain of function cells. The perpendicular black lines originating from the centrosomes and the center of the spindle midzone were used to calculate spindle angle shown in yellow and plotted as a histogram. h, DNA (blue) and HEC1 (red) were confocal imaged at M phase in OGT/OGA gain of function cells. Average width of HEC1 staining was quantified using ImageJ (mean ± S.E., replicate number (n) : nControl = 21, nOGT = 47, nOGA = 50, *, p < 0.005 between Control versus OGT/OGA). i, DNA (blue), α-tubulin (green), and HEC1 (red) were confocal imaged at M phase in TMG-treated cells. j, Western blot of HEC1 and actin in OGT/OGA gain of function and TMG-treated cells.

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