Overview

  • Product name
    Anti-Ki67 antibody [SP6]
    See all Ki67 primary antibodies
  • Description
    Rabbit monoclonal [SP6] to Ki67
  • Host species
    Rabbit
  • Specificity

    Mouse and rat species are recommended based on IHC results, we do not guarantee western blot for mouse and rat.

  • 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

      

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

Mouse and rat species are recommended based on IHC results, we do not guarantee western blot for mouse and rat.

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) »
  • Baechler SA  et al. The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis. Nat Commun 10:83 (2019). Read more (PubMed: 30622257) »
See all 724 Publications for this product

Customer reviews and Q&As

1-10 of 18 Q&A

Answer

Either ab16667 or ab27619 would work for your chosen application and species.

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Question
Answer

This antibody is a tissue culture supernatent and therefore is not purified hence we have not determined its concentration.

However, typically the concentration of tissue culture supernatants is between 10-50 ug/ml so we would expect ab16667's concentration to be within this range.

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Answer

ab16667 is cultured in serum free medium.

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Question
Answer

Since this antibody is from a tissue culture supernatant, with other proteins present, the concentration of the antibody is unknown. The typical total antibody concentration in tissue culture supernatants ranges from 1 to 3 mg/ml, with the specific antibody concentration ranging from 10-50 ug/ml.

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Answer

We have heard from customer Abreviews that ab16667 works well in frozen tissues when fixed with acetone:

https://www.abcam.com/index.html?datasheet=16667&tab=abreviews&intabreviewid=28590

https://www.abcam.com/index.html?datasheet=16667&tab=abreviews&intabreviewid=28592

I do not see any Abreviews using methanol, but it does seem that acetone should work well. Also, I believe this article cites the usage of acetone fixed tissues with this antibody:

http://www.ncbi.nlm.nih.gov/pubmed/20215534?dopt=Abstract

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Question
Answer

The buffers for these antibodies do contain BSA as follows:

Ab16667 – 1% BSA
Ab27619 – 0.5% BSA

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Question
Answer

Yes, you can use the goat anti-rabbit HRP secondary with this product for IHC. We do have two DAB kits, ab 94665 and ab64238 which are only different in size (the ab64238 is larger). The kit is just two solutions that you mix together and apply to your slides.

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Question
Answer

The Ki67 antibody ab16667 was diluted in TBST for Western blot.

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Answer

I can confirm that ab16667 Anti-Ki67 antibody [SP6] antibody is sold as tissue culture supernatant. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet. Antibody concentration is usually determined by protein assay, and serum / ascites / tissue culture supernatant will contain a lot of other proteins, which means the antibody quantification would not be accurate.

I can confirm that for tissue culture supernatant, concentration of antibody is known to very between 1 - 3 mg/ml

The isotype for this product is Rabbit IgG.

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Question
Answer

Rabbit immunoglobulins are just noted as IgG. None of our rabbit antibodies have an isotype associated with them.

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1-10 of 18 Q&A

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