Anti-Ki-67 antibody ValidAbTM
(HB7052)
Product overview
| Name | Anti-Ki-67 antibody ValidAbTM |
| Host | Mouse |
| Clonality | Monoclonal |
| Target | Ki-67 |
| Description | Antibody to Ki-67 - a widely used marker of proliferating cells. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. Ki-67 expression in proliferating HEK293T cells
HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SPE confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective and 405nm (24.5% power, PMT: 688V gain) and 488nm (29.0% power, PMT: 775V gain) lasers. Images were captured as a stack (0.23µm z-spacing) before being deconvolved with Huygens professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Ki-67 expression is inhibited in serum starved cells but rescued with 20% FBS incubation.
Serum starvation inhibits the ability of HEK293T cells to proliferate therefore inhibits Ki-67 expression. Through stimulation with 20% FBS this then strongly promotes proliferation therefore driving high expression of Ki-67. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Following this all coverslips were then fixed with ice-cold 4% paraformaldehyde in PBS. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 550 (Thermofisher, 84540) conjugated secondary antibody (1:300 dilution) and FITC Phalloidin (HB0814, 1:500 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a DFC365FX monochrome digital camera. All images were captured using a 40x objective and the same exposure settings of 73.9ms (DAPI), 73ms (FITC Phalloidin) and 107.2ms (Dy550). Images were captured as a stack (0.53µm z-spacing) before being deconvolved in Huygens Professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of HB7052 staining in HEK293T cells.
HB7502 produces reliable Ki-67 staining at concentrations as low as 1:4,000 (0.25µg/ml). Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at concentrations ranging from 2µg/ml (1:500 dilution) to 0.25µg/ml (1:4,000 dilution). This was followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. Images were captured as a z-stack using a 100x objective with the following exposures:
- 1:500 – DAPI: 4.9ms, L5: 20ms
- 1:1,000 – DAPI: 4.9ms, L5: 10ms
- 1:2,000 – DAPI: 7.9ms, L5: 61ms
- 1:4,000 – DAPI: 6ms, L5: 15.2ms
Figure 4. Ki-67 expression in proliferating HEK293T cells
HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (1.1% power, HyD: 10% gain). Images were captured as a stack (0.38µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Large population of Ki-67 expressing HEK293T cells in culture stained with HB7052.
HB7052 reveals proliferating cells within a large population of HEK293T cells grown in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. The image was captured as a tilescan and z-stack (0.3µm spacing) using a 40x objective, DAPI (4.9ms exposure) and L5 (9.1ms exposure) filters. Images were combined in LASX, deconvolved in Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Ki-67 expression is rescued by 20% FBS stimulation in serum starved HEK293T cells.
Serum starved cells (SS) express only degradation products of Ki-67 whereas 20% FBS stimulation (SS + Stim) induces new Ki-67 expression as seen by the presence of higher molecular weight bands. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Cells were subsequently lysed in Laemmli sample buffer. Rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Samples were loaded (20µg / lane) onto a 4-20% acrylamide gel alongside a protein ladder (NEB Prestained) before being run at 150V for 50 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7052 at a 1:500 dilution (2µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualized again using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure).
Figure 7. Ki-67 expression in the nucleus of a single HEK293T cell revealed with HB7052
HB7052 reveals the subcellular expression of Ki-67 in the nucleus of a proliferating HEK293T cell. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (3.1% power, HyD: 52.4% gain). Images were captured as a stack (0.12µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Ki-67 expression in proliferating HEK293T cells
HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SPE confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective and 405nm (24.5% power, PMT: 688V gain) and 488nm (29.0% power, PMT: 775V gain) lasers. Images were captured as a stack (0.23µm z-spacing) before being deconvolved with Huygens professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Ki-67 expression is inhibited in serum starved cells but rescued with 20% FBS incubation.
Serum starvation inhibits the ability of HEK293T cells to proliferate therefore inhibits Ki-67 expression. Through stimulation with 20% FBS this then strongly promotes proliferation therefore driving high expression of Ki-67. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Following this all coverslips were then fixed with ice-cold 4% paraformaldehyde in PBS. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 550 (Thermofisher, 84540) conjugated secondary antibody (1:300 dilution) and FITC Phalloidin (HB0814, 1:500 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a DFC365FX monochrome digital camera. All images were captured using a 40x objective and the same exposure settings of 73.9ms (DAPI), 73ms (FITC Phalloidin) and 107.2ms (Dy550). Images were captured as a stack (0.53µm z-spacing) before being deconvolved in Huygens Professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of HB7052 staining in HEK293T cells.
HB7502 produces reliable Ki-67 staining at concentrations as low as 1:4,000 (0.25µg/ml). Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at concentrations ranging from 2µg/ml (1:500 dilution) to 0.25µg/ml (1:4,000 dilution). This was followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. Images were captured as a z-stack using a 100x objective with the following exposures:
- 1:500 – DAPI: 4.9ms, L5: 20ms
- 1:1,000 – DAPI: 4.9ms, L5: 10ms
- 1:2,000 – DAPI: 7.9ms, L5: 61ms
- 1:4,000 – DAPI: 6ms, L5: 15.2ms
Figure 4. Ki-67 expression in proliferating HEK293T cells
HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (1.1% power, HyD: 10% gain). Images were captured as a stack (0.38µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Large population of Ki-67 expressing HEK293T cells in culture stained with HB7052.
HB7052 reveals proliferating cells within a large population of HEK293T cells grown in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. The image was captured as a tilescan and z-stack (0.3µm spacing) using a 40x objective, DAPI (4.9ms exposure) and L5 (9.1ms exposure) filters. Images were combined in LASX, deconvolved in Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Ki-67 expression is rescued by 20% FBS stimulation in serum starved HEK293T cells.
Serum starved cells (SS) express only degradation products of Ki-67 whereas 20% FBS stimulation (SS + Stim) induces new Ki-67 expression as seen by the presence of higher molecular weight bands. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Cells were subsequently lysed in Laemmli sample buffer. Rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Samples were loaded (20µg / lane) onto a 4-20% acrylamide gel alongside a protein ladder (NEB Prestained) before being run at 150V for 50 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7052 at a 1:500 dilution (2µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualized again using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure).
Figure 7. Ki-67 expression in the nucleus of a single HEK293T cell revealed with HB7052
HB7052 reveals the subcellular expression of Ki-67 in the nucleus of a proliferating HEK293T cell. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (3.1% power, HyD: 52.4% gain). Images were captured as a stack (0.12µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | Nuclei derived from the L428 Hodgkin Lymphoma cell line |
| Clone number | Ki-67 |
| Isotype | IgG1 |
| Purification | Protein A affinity chromatography |
| Concentration | 1mg/ml |
| Formulation | Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant albumin |
| Predicted species reactivity | Human, Cow |
| Tested species reactivity | Human |
Tested applications
| Applications | ICC, WB |
| Western blot optimal concentration | 2μg/ml (1:500 dilution) as tested in cultured HEK293T cells. |
| ICC optimal concentration | 0.5μg/ml (1:2,000 dilution) as tested in cultured HEK293T cells. |
| Positive control | All proliferating cells express Ki-67. An easy positive control is using any standard cell line under normal culturing conditions. |
| Negative control | Cells in G0 do not express Ki-67. An easy negative control is to serum starve cells by growing in serum free media to arrest the cell cycle and inhibit Ki-67 expression. |
| Open data link | Please follow this link to OSF |
Target information
| Other names | Proliferation marker protein Ki-67, Antigen identified by monoclonal antibody Ki-67, MKI67 |
| UniProt ID | P46013 |
| Gene name | MKI67 |
| NCBI full gene name | marker of proliferation Ki-67 |
| Entrez gene ID |
| Amino acids | 3,256 (358.7kDa) |
| Isoforms | Ki-67 has two isoforms produced by differential splicing:
|
| Expression | Ki-67 is found in all proliferating cells. |
| Subcellular expression | Ki-67 is expressed in the nucleus where its localisation changes by cell cycle phase but is predominantly found in association with DNA. |
| Processing | Ki-67 is not subject to any processing to form the active conformation. |
| Post translational modifications | Ki-67 is subject to phosphorylation on many of its serine, threonine and tyrosine residues alongside forming cross-links with SUMO1 and SUMO2 at multiple residues. |
| Homology (compared to human) | Mouse and rat Ki-67 show low homology with human Ki-67 with homology scores of 42.7% and 43.4% homology respectively. Due to this low homology HB7052 does not react with mouse and rat Ki-67. |
| Similar proteins | There are no proteins with significant homology to Ki-67 |
Storage & Handling
| Storage instructions | -20°C |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Anti-Ki-67 antibody ValidAbTM
References are publications that support the biological activity of the product
-
Ki-67 gene expression.
Uxa S et al (2021) Cell death and differentiation 28 : 3357-3370 -
Ki-67 protein as a tumour proliferation marker.
Menon SS et al (2019) Clinica chimica acta; international journal of clinical chemistry 491 : 39-45 -
Ki-67: more than a proliferation marker.
Sun X et al (2018) Chromosoma 127 : 175-186 -
Ki67 is a promising molecular target in the diagnosis of cancer (review).
Li LT et al (2015) Molecular medicine reports 11 : 1566-72 -
The Ki-67 protein: from the known and the unknown.
Scholzen T et al (2000) Journal of cellular physiology 182 : 311-22
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