Validated using a knockout cell line
Recombinant
RabMAb

Anti-Ki67 antibody [EPR3610] (ab92742)

Overview

  • Product name
    Anti-Ki67 antibody [EPR3610]
    See all Ki67 primary antibodies
  • Description
    Rabbit monoclonal [EPR3610] to Ki67
  • Tested applications
    Suitable for: IHC-Fr, WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
    Does not react with: Mouse, Rat
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human Ki67 aa 1050-1150.

  • Positive control
    • WB: HeLa and ramos cell lysates. IHC-P: Human tonsil, colon, ovarian carcinoma, squamous cell carcinoma of cervix and colonic adenocarcinoma tissues. ICC/IF: HeLa, HT-29 cells, HAP1 cells. Flow Cyt: Ramos cells.
  • General notes

    This product is not suitable for xenograft experiments. For further information please contact our Customer Services team.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab92742 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
IHC-Fr Use at an assay dependent concentration.
WB 1/5000. Predicted molecular weight: 359 kDa.

For unpurified use at 1/500 - 1/1000.

IHC-P 1/500 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

For unpurified use at 1/500 - 1/1000.

Flow Cyt 1/100 - 1/150.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    Required to maintain individual mitotic chromosomes dispersed in the cytoplasm following nuclear envelope disassembly (PubMed:27362226). Associates with the surface of the mitotic chromosome, the perichromosomal layer, and covers a substantial fraction of the chromosome surface (PubMed:27362226). Prevents chromosomes from collapsing into a single chromatin mass by forming a steric and electrostatic charge barrier: the protein has a high net electrical charge and acts as a surfactant, dispersing chromosomes and enabling independent chromosome motility (PubMed:27362226). Binds DNA, with a preference for supercoiled DNA and AT-rich DNA (PubMed:10878551). Does not contribute to the internal structure of mitotic chromosomes (By similarity). May play a role in chromatin organization (PubMed:24867636). It is however unclear whether it plays a direct role in chromatin organization or whether it is an indirect consequence of its function in maintaining mitotic chromosomes dispersed.
  • Sequence similarities
    Contains 1 FHA domain.
    Contains 16 K167R repeats.
    Contains 1 PP1-binding domain.
  • Developmental stage
    Expression occurs preferentially during late G1, S, G2 and M phases of the cell cycle, while in cells in G0 phase the antigen cannot be detected (at protein level) (PubMed:6206131). Present at highest level in G2 phase and during mitosis (at protein level). In interphase, forms fiber-like structures in fibrillarin-deficient regions surrounding nucleoli (PubMed:2674163, PubMed:8799815).
  • Post-translational
    modifications
    Phosphorylated. Hyperphosphorylated in mitosis (PubMed:10502411, PubMed:10653604). Hyperphosphorylated form does not bind DNA.
  • Cellular localization
    Chromosome. Nucleus. Nucleus, nucleolus. Associates with the surface of the mitotic chromosome, the perichromosomal layer, and covers a substantial fraction of the mitotic chromosome surface (PubMed:27362226). Associates with satellite DNA in G1 phase (PubMed:9510506). Binds tightly to chromatin in interphase, chromatin-binding decreases in mitosis when it associates with the surface of the condensed chromosomes (PubMed:15896774, PubMed:22002106). Predominantly localized in the G1 phase in the perinucleolar region, in the later phases it is also detected throughout the nuclear interior, being predominantly localized in the nuclear matrix (PubMed:22002106).
  • Information by UniProt
  • Database links
  • Alternative names
    • Antigen identified by monoclonal antibody Ki 67 antibody
    • Antigen identified by monoclonal antibody Ki-67 antibody
    • Antigen KI-67 antibody
    • Antigen KI67 antibody
    • Antigen Ki67 antibody
    • KI67_HUMAN antibody
    • KIA antibody
    • Marker of proliferation Ki-67 antibody
    • MIB 1 antibody
    • MIB antibody
    • MKI67 antibody
    • PPP1R105 antibody
    • Proliferation marker protein Ki-67 antibody
    • Proliferation related Ki 67 antigen antibody
    • Protein phosphatase 1 regulatory subunit 105 antibody
    • RP11-380J17.2 antibody
    see all

Images

  • ab92742 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab92742 at 1µg/ml and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling Ki67 with unpurified ab92742.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human squamous cell carcinoma of cervix tissue labelling Ki67 with purified ab92742 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • ab92742 staining Ki67 in human adenocarcinoma cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS and blocked with 5% serum for 1 hour at 21°C. Samples were incubated with primary antibody (1/1000) for 12 hours at 4°C. A CY3® conjugated donkey anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

    See Abreview

  • Anti-Ki67 antibody [EPR3610] (ab92742) at 1/5000 dilution (purified) + Ramos cell lysate at 20 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

    Predicted band size : 359 kDa
    Observed band size : 395 kDa (why is the actual band size different from the predicted?)

    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunocytochemistry/Immunofluorescence analysis of HT-29 cells labelling Ki67 with purified ab92742 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.

    Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • Flow Cytometry analysis of Ramos cells labelling Ki67 with purified ab92742 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • Overlay histogram showing Ramos cells stained with unpurified ab92742 (red line). The cells were fixed with 80% 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 (unpurified ab92742, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labelling Ki67 with unpurified ab92742.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal colon tissue labelling Ki67 with unpurified ab92742.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Ki67 with unpurified ab92742.

  • Unpurified ab92742 staining Ki67 in human tonsil tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized and blocked with 4% serum + 1% BSA for 30 minutes at 22°C. Samples were incubated with primary antibody (1/500 in blocking buffer) for 18 hours at 4°C. An Alexa Fluor® 568-conjugated donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody. Magnification: 20X. Counterstained with DAPI.

    See Abreview

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Ki67 with unpurified ab92742 at a dilution of 1/250.

  • Anti-Ki67 antibody [EPR3610] (ab92742) at 1/500 dilution (unpurified) + HeLa cell lysate at 10 µg

    Secondary
    HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

    Predicted band size : 359 kDa
    Observed band size : 395 kDa (why is the actual band size different from the predicted?)

References

This product has been referenced in:
  • Liu W  et al. BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis. Nat Commun 8:14182 (2017). ICC/IF ; Mouse . Read more (PubMed: 28128212) »
  • McGill TJ  et al. Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation. Transl Vis Sci Technol 6:17 (2017). IHC-P ; Rat . Read more (PubMed: 28626601) »

See all 18 Publications for this product

Customer reviews and Q&As

Application
Immunocytochemistry/ Immunofluorescence
Sample
Chicken Cell (human HCT116 in Chicken CAM)
Permeabilization
Yes - 0,5% Triton X-100
Specification
human HCT116 in Chicken CAM
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Nov 24 2017

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (brain organoid)
Permeabilization
Yes - PBS-Triton 0.5%
Specification
brain organoid
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Kalina Draganova

Verified customer

Submitted Sep 11 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Adenocarcinoma cells)
Permeabilization
Yes - 0.1% TX-100
Specification
Adenocarcinoma cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 13 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (skin tumor)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM Sodium Citrate buffer pH6
Permeabilization
No
Specification
skin tumor
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 21 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Marmoset (common) Tissue sections (Colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Colon
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Formaldehyde
Username

Mr. Carl Hobbs

Verified customer

Submitted Sep 25 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (Colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Colon
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Formaldehyde
Username

Mr. Carl Hobbs

Verified customer

Submitted Aug 19 2015

Application
Flow Cytometry
Sample
Human Cell (Differentiated hNSCs)
Permeabilization
Yes - 0.25% Triton X-100 in DPBS
Gating Strategy
Undifferentiated Stem Cells (white)
Specification
Differentiated hNSCs
Preparation
Cell harvesting/tissue preparation method: Accutase
Sample buffer: PBS
Fixation
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 15 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tonsil)
Antigen retrieval step
Heat mediated
Permeabilization
No
Specification
Tonsil
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Formaldehyde
Username

Mr. Carl Hobbs

Verified customer

Submitted Jun 18 2015

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Human Glioma into mouse brain)
Permeabilization
Yes - Triton X-100
Specification
Human Glioma into mouse brain
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 27 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample
Human Cell (Breast cancer)
Specification
Breast cancer
Permeabilization
Yes - Triton X-100
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 09 2014

1-10 of 13 Abreviews or Q&A

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