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).

All lanes : Anti-HEC1/HEC antibody [9G3] (ab3613) at 1/1000 dilution

Lane 1 : 293T whole cell lysate
Lane 2 : A431 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : HeLa whole cell lysate
Lane 5 : HeLa nuclear extract

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Mouse IgG antibody (HRP) at 1/5000 dilution

Developed using the ECL technique.

Predicted band size: 74 kDa

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. 

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.

See Abreview

HEC1/HEC was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to HEC1/HEC and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab3613.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: Band: 76kDa: HEC1/HEC.

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.

Anti-HEC1/HEC antibody [9G3] (ab3613) at 1/1000 dilution (4? , O/N) + HeLa cell lysate at 30 µg

Secondary
Mouse IgG antibody (HRP) at room temperature for 1 hour at 1/5000 dilution

Predicted band size: 74 kDa

Running conditions: run at 80V for 15 minutes then at 140V for 40 minutes

Transfer conditions: Semi-dry at 18 V for 60min (NC membrane)

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

Washing conditions: 5 ml TBST, 4 x 5min

Exposure system used: Trident plus Western HRP Substrate

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.

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.