Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Ki67 antibody [EPR3610] - BSA and Azide free (ab209897)

Overview

  • Product name

    Anti-Ki67 antibody [EPR3610] - BSA and Azide free
    See all Ki67 primary antibodies
  • Description

    Rabbit monoclonal [EPR3610] to Ki67 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, IHC-Fr, Flow Cyt, IHC-P, WBmore details
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat
  • Immunogen

    Synthetic peptide within Human Ki67 aa 1050-1150. The exact sequence is proprietary.

  • 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, KI67-HAP1 cells (WT and KO). Flow Cyt: Ramos cells.
  • General notes

    ab209897 is the carrier-free version of ab92742 This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Ab209897 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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 ab209897 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
ICC/IF Use a concentration of 1 µg/ml.
IHC-Fr Use at an assay dependent concentration.
Flow Cyt 1/100 - 1/150.

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

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

See IHC antigen retrieval protocols.

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

WB 1/5000. Predicted molecular weight: 359 kDa.

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

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

  • Comparison between RNApII-S2P-/low cells and Ki-67- cells

    a: Regulation of Ki-67 and RNApII-S2P during proliferation and quiescence in T98G glioblastoma cells. T98G cells were grown in culture medium containing 10% (v/v) fetal bovine serum (FBS), were induced to become quiescent by serum starvation in medium supplemented with 0.5% (v/v) FBS for 14 days, and then were re-stimulated by being split 1:5 into new medium containing 10% (v/v) FBS and cultured for 3 days. The cells were detached from dishes with trypsin-EDTA solution, fixed in 10% (v/v) neutral buffered formalin, and centrifuged. Paraffin sections of the pellet were cut, and expression of Ki-67 and RNApII-S2P was examined by single (brown; a1, a2, a4, a5, a7, a8) or double immunostaining (Ki-67, brown; RNApII-S2P, red; a3, a6, a9). Hematoxylin (blue) was used as a nuclear stain. Ki-67- RNApII-S2P-/low cells (blue cells in the double stained sections) emerged only in the quiescent condition (a6, arrows). Scale bar, 10 μm. b: Single-color immunostaining for Ki-67 (b1) and RNApII-S2P (b2) in serial sections of glioblastoma tissue. Ki-67- tumor cells were frequently found, whereas only a few RNApII-S2P-/low cells (arrows) were observed around necrotic area. N, necrotic area; V, blood vessels. Scale bars, 50 μm.

    Ki67 detected using ab92742.

    (From Figure S2 of Ishii et al)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Chromogenic triple immunostaining for estrogen receptor (ER), progesterone receptor (PgR), and Ki-67 in breast cancer tissue to verify the triple immunostaining detection method.

    Panel e: ER+ PgR- Ki-67- cells were stained red (short arrow), ER- PgR+ Ki-67- cells were stained blue (black arrowhead), ER+ PgR+ Ki-67- cells were stained purple (long arrow), and Ki-67+ cells were stained brown (white arrowhead). These colors are easily distinguishable. Scale bars, 25 μm.

    Deparaffinized sections were pretreated for antigen retrieval by boiling in antigen retrieval solution, pH 9. Sections were incubated with rabbit monoclonal antibody against Ki67 ab92742 at a 1/1000 dilution. After the reaction with (HRP)-conjugated secondary antibodies color was developed with (DAB) and sections were counterstained with hematoxylin.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human squamous cell carcinoma of cervix tissue labeling 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). Counterstained with hematoxylin.

    Negative control using PBS instead of primary antibody (inset).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Immunocytochemistry/Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma cell line) cells labeling 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Flow Cytometry analysis of Ramos (Human Burkitt's lymphoma cell line) cells lablling Ki67 with purified ab92742 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. An FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • 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 labeled in blue with DAPI.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Overlay histogram showing Ramos (Human Burkitt's lymphoma cell line) 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Ki67 with unpurified ab92742 at a dilution of 1/250.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

  • ab209897 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 ab209897 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.

  • Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Ki67 with ab209897 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Nucleus staining on human tonsil.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Human bladder cancer labeling Ki67 with ab209897 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Nucleus staining on human bladderr cancer.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

References

This product has been referenced in:

  • He Q  et al. Methionine Sulfoxide Reductase B1 Regulates Hepatocellular Carcinoma Cell Proliferation and Invasion via the Mitogen-Activated Protein Kinase Pathway and Epithelial-Mesenchymal Transition. Oxid Med Cell Longev 2018:5287971 (2018). Human . Read more (PubMed: 29861830) »
  • Zhang J  et al. SOX4 inhibits GBM cell growth and induces G0/G1 cell cycle arrest through Akt-p53 axis. BMC Neurol 14:207 (2014). WB ; Human . Read more (PubMed: 25366337) »
See all 4 Publications for this product

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