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Anti-Ki67 Antibody Picoband™ (monoclonal, 5E12)

Figure 1. IHC analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 2. IHC analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human cervical cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 3. IHC analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 4. IHC analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 5. IHC analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 6. IF analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 7. IF analysis of Ki67 using anti-Ki67 antibody (M00254-8).
Ki67 was detected in paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 µg/mL mouse anti-Ki67 Antibody (M00254-8) overnight at 4°C. Biotin conjugated goat anti-mouse IgG (BA1001) was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using DyLight®488 Conjugated Avidin (BA1128). The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 8. Flow Cytometry analysis of Jurkat cells using anti-Ki67 antibody (M00254-8).
Overlay histogram showing Jurkat cells stained with M00254-8 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Ki67 Antibody (M00254-8, 1 µg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10 µg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

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BACKGROUND

Product Information
Application Applications Legend: WB=Western Blot IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin-embedded Sections) IHC-F=Immunohistochemistry (Frozen Sections) IF=Immunofluorescence FC=Flow Cytopmetry IC=Immunochemistry ICC=Immunocytochemistry E=ELISA IP=Immunoprecipitation DB=Dot Blot CHIP=Chromatin Immunoprecipitation FA=Fluorescence Assay IEM=Immuno electron microscopy EIA=Enzyme Immunoassay	IHC, IF, ICC, FC
Primary Accession	P46013
Host	Mouse
Isotype	Mouse IgG2b
Reactivity	Human
Clonality	Monoclonal
Format	Lyophilized
Description	Anti-Ki67 Antibody Picoband™ (monoclonal, 5E12) . Tested in Flow Cytometry, IF, IHC, ICC applications. This antibody reacts with Human.
Reconstitution	Add 0.2ml of distilled water will yield a concentration of 500ug/ml.

Additional Information
Gene ID	4288
Other Names	Proliferation marker protein Ki-67, Antigen identified by monoclonal antibody Ki-67, Antigen KI-67, Antigen Ki67, MKI67 (HGNC:7107)
Calculated MW	358694 Da
Application Details	Immunohistochemistry (Paraffin-embedded Section), 2-5 µg/ml, Human Immunocytochemistry/Immunofluorescence, 5 µg/ml, Human Immunofluorescence, 5 µg/ml, Human Flow Cytometry, 1-3 µg/1x10^6 cells, Human
Contents	Each vial contains 4mg Trehalose, 0.9mg NaCl and 0.2mg Na2HPO4.
Clone Names	Clone: 5E12
Immunogen	E. coli-derived human Ki67 recombinant protein (Position: K2860-I3256).
Purification	Immunogen affinity purified.
Storage	Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.

Protein Information
Name	MKI67 (HGNC:7107)
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 (Probable).
Cellular Location	Chromosome. Nucleus. Nucleus, nucleolus Note=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).

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Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol

Background

Ki-67 (Proliferation-related Ki-67 antigen), also known as MKI67 or KIA, is a protein that in humans is encoded by the MKI67 gene. From study of a panel of human-rodent somatic cell hybrids, it has been demonstrated that a gene involved in the expression of the MKI67 antigen is located on chromosome 10. By in situ hybridization, Fonatsch et al. (1991) regionalized the MKI67 gene to chromosome 10q25-qter. By FISH, Traut et al. (1998) mapped the mouse Mki67 gene to chromosome 7F3-F5. Antigen KI-67 is a nuclear protein that is associated with and may be necessary for cellular proliferation. Furthermore it is associated with ribosomal RNA transcription. Inactivation of antigen KI-67 leads to inhibition of ribosomal RNA synthesis.

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