使用敲除细胞株进行验证RabMAb

Anti-Ki67抗体[EPR3610] (ab92742)

概述

  • 产品名称Anti-Ki67抗体[EPR3610]
    参阅全部 Ki67 一抗
  • 描述
    兔单克隆抗体[EPR3610] to Ki67
  • 经测试应用适用于: IHC-Fr, WB, IHC-P, Flow Cyt, ICC/IFmore details
  • 种属反应性
    与反应: Human
    不与反应: Mouse, Rat
  • 免疫原

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

  • 阳性对照
    • 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.
  • 常规说明

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

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

    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.

性能

应用

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.

应用 Ab评论 说明
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.

靶标

  • 功能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.
  • 序列相似性Contains 1 FHA domain.
    Contains 16 K167R repeats.
    Contains 1 PP1-binding domain.
  • 发展阶段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).
  • 翻译后修饰Phosphorylated. Hyperphosphorylated in mitosis (PubMed:10502411, PubMed:10653604). Hyperphosphorylated form does not bind DNA.
  • 细胞定位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
  • 数据库链接
  • 别名
    • 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

Anti-Ki67 antibody [EPR3610] 图像

  • 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.

  • 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?)

Anti-Ki67 antibody [EPR3610] (ab92742)参考文献

This product has been referenced in:
  • Qin Q  et al. miR-134 inhibits non-small cell lung cancer growth by targeting the epidermal growth factor receptor. J Cell Mol Med N/A:N/A (2016). IHC ; Human . Read more (PubMed: 27241841) »
  • Zhang HJ  et al. Twist2 promotes kidney cancer cell proliferation and invasion by regulating ITGA6 and CD44 expression in the ECM-receptor interaction pathway. Onco Targets Ther 9:1801-12 (2016). WB ; Human . Read more (PubMed: 27099513) »

See all 13 Publications for this product

Product Wall

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

提交于 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

提交于 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

提交于 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

提交于 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

提交于 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

提交于 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

提交于 Jun 09 2014

Application Immunohistochemistry (Frozen sections)
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 4% · Temperature: 22°C
Sample Human Tissue sections (Tonsil)
Specification Tonsil
Permeabilization Yes - 0.5%
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 May 28 2014

I tried to find the mouse and human specific skin cell markers, regrettably I couldn't find enough information about protein that are specific to human or mouse cells. I am sure further publication search will be helpful in answering this question. I w...

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ab92742 has indeed been tested in western blot on mouse and rat samples and gave a negative result.

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