Overview

  • Product nameAnti-Ki67 antibody
    See all Ki67 primary antibodies
  • Description
    Rabbit polyclonal to Ki67
  • SpecificityAb15580 is batch tested in ICC/IHC. Some customers have successfully used ab15580 in Western Blot. Higher molecular weight proteins like ki67 may be more difficult to detect in WB. We recommend several potential optimisation steps: loading higher amounts of protein (20 ug and above), using lower percentage gels and/or Tris-Acetate gels, increasing antioxidant to maintain protein reduction, decreasing methanol and increasing SDS in the transfer buffer and increasing time and voltage of transfer. Larger proteins can be subject to degradation more than smaller proteins so lower molecular weight bands may be present. For further information or support please contact our Scientific Support Team.
  • Tested applicationsSuitable for: IHC - Wholemount, IHC-P, IHC-FrFl, Flow Cyt, IHC-Fr, ICC/IF, ICC, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Horse, Cow, Dog, Human, Pig, Indian Muntjac, Monkey, Chinese Hamster, Marmoset (common), Syrian Hamster
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1200 - 1300 of Human Ki67.

    (Peptide available as ab15581.)

  • Positive control
    • ICC-IF: Ki67 wildtype and knockout HAP1 cells IF: mouse (P0) olfactory bulb, MEF1 IHC-Fr: mouse P7 brain sections IHC-P: mouse spleen

Properties

Applications

Our Abpromise guarantee covers the use of ab15580 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 - Wholemount Use at an assay dependent concentration.
IHC-P Use a concentration of 0.1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-FrFl 1/500. (see Abreview)
Flow Cyt 1/100.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-Fr 1/100 - 1/1000.
ICC/IF Use a concentration of 1 µg/ml.
ICC Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration.

Target

  • FunctionThought to be required for maintaining cell proliferation.
  • Sequence similaritiesContains 1 FHA domain.
  • Developmental stageExpression of this antigen 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.
  • Cellular localizationNucleus. Nucleus, nucleolus. Chromosome. 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. In mitosis, it is present on all chromosomes.
  • Information by UniProt
  • Database links
  • Alternative names
    • Antigen identified by monoclonal antibody Ki 67 antibody
    • Antigen KI-67 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 related Ki 67 antigen antibody
    • Protein phosphatase 1 regulatory subunit 105 antibody
    • RP11-380J17.2 antibody
    see all

Anti-Ki67 antibody images

  • ab15580 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 ab15580 at 1μg/ml concentration 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.

  • IHC image of ab15580 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab15580, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  • ab15580 staining Ki67 - Proliferation Marker in Human PBMCs by Flow Cytometry. Cells were isolated by Ficoll density separation, fixed in paraformaldehyde and permeabilized in 0.1% Saponin + PBS. The sample was incubated with the primary antibody (1/100 in PBS + 1% FCS and 0.09% sodium azide) for 1 hour at 4°C. A phycoerythrin-conjugated Goat anti-rabbit IgG (1/100) was used as the secondary antibody.

    Gating Strategy: Lymphocytes and Monocytes

    1 = isotype; 2 = PBMCs untreated; 3 = PBMCs treated with PHA.

    See Abreview

  • Immunostaining for Rabbit polyclonal to Ki67 - Proliferation Marker (ab15580) on Mouse P7 brain (frozen) sections. Sections were fixed with paraformaldehyde, neither a blocking nor antigen retrieval steps were included in this protocol. Secondary antibody used was Rabbit IgG antibody (ab7082; 1/250).
  • IHC image of Ki67 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab15580, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Fluorescent confocal microscopy (20x) of mouse (P0) olfactory bulb, outer glomeruli layer, showing Ki67 immunoreactivity (ab15580; 1/1000; overnight at RT, 0.25% TX-100 no blocking step) using a secondary goat anti-rabbit fluorescent antibody (Alexa Fluor 488;1/300 2h at RT.

  • ab15580 staining Ki67 in SK-N-SH cells treated with NADA (N-Arachidonyldopamine) (ab120099), by ICC/IF. Decrease in Ki67 expression correlates with increased concentration of NADA (N-Arachidonyldopamine), as described in literature.
    The cells were incubated at 37°C for 10 minutes in media containing different concentrations of ab120099 (NADA (N-Arachidonyldopamine)) in ethanol, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab15580 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
  • SK-N-SH cells were permitted to grow to confluency, then serum starved for 48 hours and predominantly driven into G0. The cells were then paraformaldehyde fixed and immunofluorescently labelled with anti-Ki67 (ab15580) at a dilution of 1/1000. The majority of the cells show little or no Ki67 staining, indicating they are in G0 arrest (red cells). Two cells however show strong nucleolar Ki67 staining indicating they are still cycling (green cells). The DNA is stained with DAPI and is shown in red. The Ki67 staining is shown in green. x 63 magnification.

    Similar results were seen with an asynchronous population of HeLa cells. The Ki67 staining was localised to the periphery of the nucleoli and throughout the nucleoplasm of proliferating cells. (This data is not shown but is available upon request).

  • ab15580 at 1/50 staining Human umbilical artery endothelial cells by ICC/IF. The tissue was paraformaldehyde fixed and blocked before permeabilization with saponin and incubation with the antibody for 16 hours. A FITC conjugated goat anti-rabbit IgG was used as the secondary. The merged image shows those cells expressing Ki67 from the total number of exponential cells.

    See Abreview

  • IHC image of ab15580 stained human skin carcinoma FFPE section. Section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30 seconds at 125°C. Section was incubated with ab15580 at a dilution of 1:200 for 1h at room temperature and detected using an HRP conjugated polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • ab15580 staining Ki67-Proliferation Maker in human colon tissue sections by IHC-P (formaldehyde-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde and blocked with 10% goat serum  for 30 minutes at 25°C. Antigen retrieval was by heat mediation in Target Retrieval Solution. Samples were incubated with primary antibody 1/5000 (TBST) for 1 hour at 25°C. 

    See Abreview

  • Immunohistochemistry analysis of Formaldehyde-fixed paraffin-embedded mouse tumour tissue sections labelling Ki67 with ab15580 at 1/2000. The secondary antibody was biotin conjugated goat polyclonal vector at a dilution of 1/250.  

    See Abreview

  • ICC/IF image of ab15580 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab15580, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab15580 staining Ki67 - Proliferation Marker in Human skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 4% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer (pH 6.0). Samples were incubated with primary antibody (5 µg/ml in blocking buffer) for 16 hours at 4°C. A Texas Red ® Goat anti-rabbit IgG polyclonal (1/100) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab15580 stained MEF1 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab15580, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References for Anti-Ki67 antibody (ab15580)

This product has been referenced in:
  • Tian LQ  et al. MicroRNA-197 inhibits cell proliferation by targeting GAB2 in glioblastoma. Mol Med Rep N/A:N/A (2016). Read more (PubMed: 27035789) »
  • Allen KM  et al. Cell proliferation is reduced in the hippocampus in schizophrenia. Aust N Z J Psychiatry 50:473-80 (2016). IHC (PFA fixed) ; Human . Read more (PubMed: 26113745) »

See all 284 Publications for this product

Product Wall

Application Western blot
Sample Human Cell lysate - whole cell (MCF-7)
Gel Running Conditions Reduced Denaturing (10)
Loading amount 20 µg
Specification MCF-7
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Aug 30 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application IHC - Wholemount
Sample Mouse Tissue (Gastric organoids)
Specification Gastric organoids
Username

Abcam user community

Verified customer

Submitted Aug 23 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Tumour)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate
Permeabilization No
Specification Tumour
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: RT°C
Fixative Formaldehyde
Username

Jim Manavis

Verified customer

Submitted Aug 16 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 (prostate cancer)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate buffer, pH6.0
Permeabilization No
Specification prostate cancer
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 29 2016

Application Immunocytochemistry/ Immunofluorescence
Sample Rat Cell (Rat prostate)
Permeabilization Yes - 0.2% Triton X-100
Specification Rat prostate
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative Acetone
Username

Dr. Armen Petrosyan

Verified customer

Submitted Jun 18 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (lungs)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate buffer pH 6.0
Specification lungs
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative Formaldehyde
Username

Mariya Farooqui

Verified customer

Submitted May 12 2016

Application Immunohistochemistry (Frozen sections)
Sample Human Tissue sections (Skin)
Permeabilization No
Specification Skin
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted May 09 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Skin)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Sodium Citrate pH 6
Permeabilization No
Specification Skin
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: RT°C
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 09 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 (Colon)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: TRIS-EDTA Buffer pH 9,0
Permeabilization Yes - Wash Buffer from Dako with Tween
Specification Colon
Fixative Paraformaldehyde
Username

Mr. Rudolf Jung

Verified customer

Submitted Apr 29 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Liver)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: TRIS-EDTA Buffer pH 9,0
Permeabilization Yes - Wash Buffer from Dako with Tween
Specification Liver
Fixative Paraformaldehyde
Username

Mr. Rudolf Jung

Verified customer

Submitted Apr 29 2016

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