Overview

  • Product nameAnti-HEC1 antibody [9G3]
    See all HEC1 primary antibodies
  • Description
    Mouse monoclonal [9G3] to HEC1
  • Tested applicationsSuitable for: Flow Cyt, IP, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human, Pig, African Green Monkey, Chinese Hamster
  • Immunogen

    Recombinant protein encoding amino acids 56-642 of human HEC 1 purified from E. coli.

  • Positive control
    • HeLa whole cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab3613 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
Flow Cyt Use 1µg for 106 cells. ab170191-Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
IP Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately 80 kDa (predicted molecular weight: 74 kDa).
ICC/IF Use at an assay dependent concentration.

Target

  • FunctionActs as a component of the essential kinetochore-associated NDC80 complex, which is required for chromosome segregation and spindle checkpoint activity. Required for kinetochore integrity and the organization of stable microtubule binding sites in the outer plate of the kinetochore.
  • Sequence similaritiesBelongs to the NDC80/HEC1 family.
  • Developmental stageExpression peaks in mitosis.
  • Post-translational
    modifications
    Phosphorylation begins in S phase of the cell cycle and peaks in mitosis. Phosphorylated by NEK2. May also be phosphorylated by AURKA and AURKB.
  • Cellular localizationNucleus. Chromosome > centromere > kinetochore. Localizes to kinetochores from late prophase to anaphase. Localizes specifically to the outer plate of the kinetochore.
  • Information by UniProt
  • Database links
  • Alternative names
    • from Entrez Gene ; antibody
    • HEC antibody
    • Highly expressed in cancer antibody
    • Highly expressed in cancer protein antibody
    • Highly expressed in cancer rich in leucine heptad repeats antibody
    • HsHec1 antibody
    • hsNDC80 antibody
    • Kinetochore associated 2 antibody
    • Kinetochore associated protein 2 antibody
    • Kinetochore protein Hec1 antibody
    • Kinetochore protein NDC80 homolog antibody
    • Kinetochore-associated protein 2 antibody
    • KNTC2 antibody
    • ndc80 antibody
    • NDC80 homolog kinetochore complex component antibody
    • NDC80 kinetochore complex component homolog antibody
    • NDC80, S. cerevisiae, homolog of antibody
    • NDC80_HUMAN antibody
    • Retinoblastoma associated protein HEC antibody
    • Retinoblastoma-associated protein HEC antibody
    • TID3 antibody
    see all

Anti-HEC1 antibody [9G3] images

  • Anti-Hec1 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 (green).
    This image was kindly supplied as part of the review submitted by Marko Kallio.


  • Predicted band size : 74 kDa
    ab3613 at a 1/1000 dilution staining ~ 80 kDa HEC 1 in Hela cell lysate (30µg per well) by Western blot. ab3613 at a 1/1000 dilution staining ~ 80 kDa HEC 1 in Hela cell lysate (30µg per well) by Western blot.
  • HEC1 was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to HEC1 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 70oC; 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.
  • 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

  • HeLA cells were stained with anti-HEC1 (ab2613; in green) and DAPI (blue) in panel 1, and with anti-HEC1(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. 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.
  • 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.

References for Anti-HEC1 antibody [9G3] (ab3613)

This product has been referenced in:
  • Beh TT  et al. Active centromere and chromosome identification in fixed cell lines. Mol Cytogenet 9:28 (2016). ICC/IF ; Human . Read more (PubMed: 27011768) »
  • Iimori M  et al. Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability. Nat Commun 7:11117 (2016). ICC/IF ; Human . Read more (PubMed: 27030108) »

See all 47 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Permeabilization Yes - PBS/0.2% Triton X-100 for 30 minutes
Specification HeLa
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 23°C
Fixative Formaldehyde
Username

Dr. Weiguo Zhang

Verified customer

Submitted Aug 07 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Sample Human Cell (HEK293)
Specification HEK293
Permeabilization Yes - triton
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 20 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
Sample Hamster Cell (Chinese Hamster Ovary, T-REx™-CHO Cell Line)
Specification Chinese Hamster Ovary, T-REx™-CHO Cell Line
Permeabilization Yes - Triton 1%
Fixative Paraformaldehyde
Username

Dr. Eleni Petsalaki

Verified customer

Submitted Oct 16 2013

In general, issues like this arise from the secondary antibody. Often the antibody is too concentrated or is reacting with something in you buffers (usually milk or BSA) Have you had the opportunity to reduce the concentration of the secondary antibody...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Colon carcinoma)
Specification Colon carcinoma
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% Triton in PHEM
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 37°C
Username

Dr. Eleni Petsalaki

Verified customer

Submitted Oct 08 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Brain (Embryonic day 17.5))
Specification Brain (Embryonic day 17.5)
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% Triton
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Aug 07 2012

Thank you for enquiry and your interest.

I can confirm that this antibody (ab3613) recognizes Mouse, Human, Pig, African Green Monkey - it has not been tested in yeast and currently we do not have antibodies which detect HEC1 in Saccharomyces...

Read More

I appreciate your cooperation and understand your concerns. It is regrettable that this product has not matched the standards of those previously.

Under our Abpromise, we guarantee that our product will work in a the manner stated on the dat...

Read More

Thank you for contacting Abcam.

I have called and left a message with you in regards to this issue. Feel free to disregard that call should this be a better forum for communicating.

I am sorry that the latest lot of this product i...

Read More

Thank you very much for your phone call. The dilution given on the datasheet is for guideline purpose only. The dilutions have to be optimized by the end user based on cells or tissue sections they use. We recommend using a range e.g. 1/500, 1/1000 and...

Read More

1-10 of 18 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"