Overview

  • Product name
    Anti-Ki67 antibody [SP6]
    See all Ki67 primary antibodies
  • Description
    Rabbit monoclonal [SP6] to Ki67
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-FoFr, ICC/IF, Flow Cyt, IHC-Fr, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Common marmoset
  • Immunogen

    Synthetic peptide within Human Ki67 aa 1200-1300. The exact sequence is proprietary.
    Database link: P46013

  • Epitope
    C-terminus
  • Positive control
    • IHC-P: Human tonsil and testis tissue. Common marmoset spleen tissue. Rat esophagus, small intestine and liver tissue. Mouse embryonic skin tissue. Transgenic mouse spinal cord tissue. Rat lymph node tissue. ICC/IF: HAP1 cells. Human cardiac stem cells. HEp-2 cells. Rat cardiomyocytes. Flow Cyt: HAP1 cells.
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab16667 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-FoFr 1/5000.

Antigen retrieval: Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min.

ICC/IF 1/250.
Flow Cyt 1/1000.

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

IHC-Fr 1/1000. A higher dilution is recommended for frozen tissues than for FFPE tissues. We suggest a starting dilution of 1/500.
WB Use at an assay dependent concentration. PubMed: 20562294
IHC-P 1/200.

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

  • ab16667 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol for 5 minutes, 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 1 hour. The cells were then incubated with ab16667 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

  • Immunostained tissue sections from control-, AT1-, and MLL-LNs

    Proliferating cells (Ki67+, brown) were seen in lymph follicles (presumably B-lymphocytes) and in para-follicular areas (presumably T-lymphocytes) in control-LNs. In AT1-LNs, the para-follicular areas dominated, and contained most of the proliferating cells. In MLL-LNs, the para-follicular regions appeared to contain less proliferating cells than in AT1-LNs.

    Frozen sections of rat lymph node tissue were stained for Ki67 using ab16667 in immunohistochemical analysis. DAB staining.

    (From Figure 10A of Strömvall et al)

  • Immunohistochemical analysis of human tonsil tissue labeling Ki-67 with ab16667 at 1/200. The HRP/AEC-staining procedure was used for detection.

  • Overlay histogram showing HAP1 wildtype (green line) and HAP1-MKI67 knockout cells (red line) stained with ab16667. The cells were fixed with  80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab16667,1/1000) for 30 min at 22°C. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 1/2000 dilution for 30 min at 22°C.
    A Rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MKI67 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
    Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
  • Effect of newborn anoxia and PD156707 on proliferation and binucleation of neonatal cardiomyocytes

    A representative image of cardiomyocytes stained with α-actinin (green), Ki-67 (red), and Hoescht (blue).

    4% paraformaldehyde-fixed, Triton X-100 permeabilized rat cardiomyocytes were stained for Ki67 using ab16667 at 1/100 dilution in ICC/IF.

    (From Figure 3A of Paradis et al)

  • ab16667 staining Ki67 (red) in transgenic mouse spinal cord tissue sections (depleted of oligodendrocytes) by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, permeablized with 0.5% Triton X-100 and blocked with 10% serum for 1 hour at 25°C; antigen retrieval was by heat mediation in 10 mM citrate buffer, pH 6, for 20 minutes at 97°C in a water bath. The sample was incubated with primary antibody (1/300 in PBS + 0.1% Triton X-100 + 1% serum) at 25°C for 16 hours. An Alexa Fluor® 594-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/700) was used as the secondary antibody. Counterstained with Iba1 (green) a marker for microglia and DAPI.

    See Abreview

  • ab16667 staining Ki67 in common marmoset spleen by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then blocked with 1% BSA for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/100. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence analysis of human cardiac stem cells labeling Ki67 with ab16667 at 1/250 dilution. Cells were fixed in paraformaldehyde and permeabilized with Triton x-100, 0.01%. Cells were blocked in BSA for 1 hour at room temperature. A polyclonal chicken anti-rabbit Alex Fluor® 488 secondary antibody was used at 1/500 dilution. 

    See Abreview

  • ab16667 staining Ki67 in rat liver tissue sections by immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 2 hours at 22°C. Samples were incubated with primary antibody (1/500 in PBS-T + 1% BSA) for 18 hours at 4°C. A biotin-conjugated goat anti-rabbit IgG monoclonal (1/2000) was used as the secondary antibody.

    See Abreview

  • ab16667 staining Ki67 in human testis by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then blocked with 1% BSA for 10 minutes at 21ºC followed by incubation with the primary antibody for 2 hours at 1/100. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

    See Abreview

  • ab16667 staining Ki67 in mouse embryonic skin tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then blocked with 1% BSA for 10 minutes at 21ºC followed by incubation with the primary antibody for 16 hours at 1/50. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

    See Abreview

  • ab16667 staining Ki67 in rat oesophagus by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then blocked with 1% BSA for 10 minutes at 21ºC followed by incubation with the primary antibody for 30 minutes at 1/100. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

    See Abreview

  • ab16667 staining Ki67 in rat small intestine tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using 10 mM citrate buffer pH 6.0. Samples were then blocked with 10% serum for 20 minutes at room temperature followed by incubation with the undiluted primary antibody for 30 minutes. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/2000 dilution.

    See Abreview

  • ab16667 staining Ki67 - Proliferation Marker in human HEp-2 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 0.25% in PBS. Samples were incubated with primary antibody (1/50 in DPBS) for 1 hour at 21°C. An Atto488-conjugated Donkey anti-rabbit polyclonal (1/50) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • He M  et al. Mesenchymal stem cells-derived IL-6 activates AMPK/mTOR signaling to inhibit the proliferation of reactive astrocytes induced by hypoxic-ischemic brain damage. Exp Neurol 311:15-32 (2019). Read more (PubMed: 30213506) »
  • Shi J  et al. Long non-coding RNA RUNX1-IT1 plays a tumour-suppressive role in colorectal cancer by inhibiting cell proliferation and migration. Cell Biochem Funct 37:11-20 (2019). Read more (PubMed: 30499136) »
See all 688 Publications for this product

Customer reviews and Q&As

91-100 of 109 Abreviews or Q&A

Question
Answer

The recommended protocol for ICC is as follows: -
1)Fix cells in 4% paraformaldehyde for 10 minutes at room temperature.
2)Permeabilize cells by incubating with 0.25% Triton X-100 in PBS for 5 min at room temperature. -
3)Block with 10% serum for 30 min at room temperature.
4)Incubate cells with primary antibody at 1:50 for 1 hour at room temperature.
5)Washes can be performed with PBS.
An alternative fixative is to use acetone: at -20oC for 5-10 minutes. This will also permeabilize so no treatment is needed. Both can be done after spinning cells, however both methods will likely destroy the epitope.

Read More

Answer

Please review the Abreview by Rudolf Lung (IHC) from 17 May 2011 for an image of the antibody used in mouse. I can confirm that ab16667 is tested and guaranteed to work in mouse and ICC/IF as well as IHC-FoFr, IHC-Fr, WB, IHC-P by our Abpromise.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Glial Tumor)
Specification
Glial Tumor
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C

Dr. R Berahovich

Verified customer

Submitted Dec 01 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Skin)
Specification
Skin
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer pH 6.0
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C

Dr. Teo Manestar-Blazic

Verified customer

Submitted Sep 13 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Rat and mouse spleens)
Specification
Rat and mouse spleens
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer, pH6.0
Permeabilization
No
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 25 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (spleen)
Specification
spleen
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TRIS-EDTA-Buffer
Permeabilization
Yes - Wash-Buffer with Tween from Dako

Mr. Rudolf Jung

Verified customer

Submitted May 17 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Small Intestine)
Specification
Small Intestine
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric buffer pH6.0
Permeabilization
No
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: Rm°C

Jing Ma

Verified customer

Submitted Mar 15 2011

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Adult rat ventricular cardiomyocytes and c-kit pos)
Specification
Adult rat ventricular cardiomyocytes and c-kit pos
Fixative
Formaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: ~22°C

Abcam user community

Verified customer

Submitted Jan 13 2011

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEp-2)
Specification
HEp-2
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100 0.25% in PBS

Mr. Peter Zentis

Verified customer

Submitted Sep 10 2010

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Liver)
Specification
Liver
Fixative
Formaldehyde
Antigen retrieval step
None
Permeabilization
No
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jun 11 2010

91-100 of 109 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up