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The antibody was purified by affinity chromatography and conjugated with Pacific Blue™ under optimal conditions. The solution is free of unconjugated Pacific Blue™ and unconjugated antibody.
Storage & Handling:
The antibody solution should be stored undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Each lot of this antibody is quality control tested by our Ki-67 staining protocol below. For immunofluorescent staining, the suggested use of this reagent is 5 µl per million cells or 5 µl per 100 µl of whole blood. It is recommended that the reagent be titrated for optimal performance for each application.
* Pacific Blue™ has a maximum emission of 455 nm when it is excited at 405 nm. Prior to using Pacific Blue™ conjugate for flow cytometric analysis, please verify your flow cytometer's capability of exciting and detecting the fluorochrome. ** Pacific Blue™ is a registered trademark of Molecular Probes, Inc. Pacific Blue™ dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.
Additional reported applications (for the relevant formats) include: immunohistochemical staining of frozen tissue sections1, Western blotting3, and immunofluorescence microscopy4.
Ki-67 Staining Protocol:
1. Prepare 70% Ethanol and chill at -20°C. 2. Prepare target cells of interest and wash 2X with PBS by centrifuge at 350xg for 5 minutes. 3. Discard supernatant and loosen the cell pellet by vortexing. 4. Add 3 ml cold 70% ethanol drop by drop to the cell pellet while vortexing. 5. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour. 6. Wash 3X with BioLegend Cell Staining Buffer and then resuspend the cells at the concentration of 0.5-10 x 106/ml. 7. Mix 100 µl cell suspension with proper fluorochrome-conjugated Ki-67 antibody and incubated at room temperature in the dark for 30 minutes. 8. Wash 2X with BioLegend Cell Staining and then resuspend in 0.5 ml cell staining buffer for flow cytometric analysis.
Application References:
1. Gerdes J, et al. 1983. Int. J. Cancer 31:13. (IHC) 2. Gerdes J, et al. 1984. J. Immunol. 133:1710. (ICFC) 3. Schluter C, et al. 1993 J. Cell Biol. 123:513. (IHC, WB) 4. Bading H, et al. 1989 Exp. Cell. Res. 185:50. (IF)
PHA-stimulated (3 days) human peripheral blood lymphocytes were fixed and permeabilized with 70% ethanol, and then stained with Ki-67 Pacific Blue™ (filled histogram) or mouse IgG1, κ Pacific Blue™ isotype control (open histogram).
Antigen Ki-67 is a nuclear protein expressed as two isoforms with molecular weights of 395 and 345 kD. Both isoforms contain one forkhead-associated domain and 16 concatenated "Ki-67 repeats," each containing the epitope recognized by the mAb Ki-67. The antigen Ki-67 interacts with Hklp2, hNIFK, and Chromobox protein homolog 1, 3, and 5. Ki-67 is required for cell proliferation and its expression is restricted to the phases G1, S, G2, and M of the cell cycle. This characteristic makes Ki-67 an excellent marker for proliferating cells and is commonly used as one of the prognostic factors in cancer studies. Ki-67 has also been used to study myocyte proliferation after myocardial infarction as well as lymphocyte proliferation during infection, and has been used in neurons of patients with different neuropathologies.
Other Names:
Antigen Ki-67
Structure:
Two isoforms with molecular weights of 395 and 345 kD, one forkhead-associated domain, 16 concatenated Ki-67 repeats, located in nucleus
Distribution:
Expressed in the phases G1, S, G2, and M of the cell cycle
Function:
Required for cell proliferation
Interaction:
Chromobox protein homolog 1, 3 and 5, Hklp2, and hNIFK
Antigen References:
1. Byeon IJ, et al. 2005. Nat. Struct. Mol. Biol. 12:987. 2. Yerushalmi R, et al. 2010. Lancet. Oncol. 11:174. 3. Beltrami AP, et al. 2001. N. Engl. J. Med. 344:1750. 4. Sachsenberg N, et al. 1998. J. Exp. Med. 187:1295. 5. Nagy Z, et al. 1997. Acta. Neuropathol. 93:294.
*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/terms). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.
This data display is provided for general comparisons between formats. Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors. If you need assistance with selecting the best format contact our expert technical support team.
Purified anti-human Ki-67
Human T leukemia cell line, Jurkat, fixed and permeabilized with 70% ethanol, then intracellularly stained with Ki-67 PE and counterstained with 7-AAD (Cat No. 420404) for DNA staining.
Resting (dashed line) or PHA-activated human peripheral blood lymphocytes (day-3, solid line) fixed and permeabilized with 70% ethanol, then intracellularly stained with Ki-67 PE.
PE anti-human Ki-67
Human T leukemia cell line, Jurkat, fixed and permeabilized with 70% ethanol, then intracellularly stained with Ki-67 PE and counterstained with 7-AAD (Cat No. 420404) for DNA staining.
Resting (dashed line) or PHA-activated human peripheral blood lymphocytes (day-3, solid line) fixed and permeabilized with 70% ethanol, then intracellularly stained with Ki-67 PE.
Brilliant Violet 421™ anti-human Ki-67
3-day PHA-stimulated (top) or non-stimulated (bottom) human peripheral blood lymphocytes were fixed with 70% cold ethanol, then stained with Ki-67 Brilliant Violet 421™ (filled histogram) or mouse IgG1 Brilliant Violet 421™ isotype control (open histogram).
Alexa Fluor® 488 anti-human Ki-67
PHA-activated human peripheral blood lymphocytes (3 days) were fixed and permeabilized with 70% ethanol, and then were stained with Ki-67 Alexa Fluor® 488 (filled histogram) or mouse IgG1, κ Alexa Fluor® 488 isotype control (open histogram).
3-day PHA-stimulated human peripheral blood lymphocytes were fixed and permeabilized with BioLegend FOXP3 buffer set, then stained with Ki-67 Alexa Fluor® 488 (filled histogram) or mouse IgG1, κ Alexa Fluor® 488 isotype control (open histogram).
Alexa Fluor® 647 anti-human Ki-67
PHA-stimulated (3 days) human peripheral blood lymphocytes were fixed and permeabilized with 70% ethanol, and then stained with Ki-67 Alexa Fluor® 647 (filled histogram) or mouse IgG1 κ Alexa Fluor® 647 isotype control (open histogram).
Pacific Blue™ anti-human Ki-67
PHA-stimulated (3 days) human peripheral blood lymphocytes were fixed and permeabilized with 70% ethanol, and then stained with Ki-67 Pacific Blue™ (filled histogram) or mouse IgG1, κ Pacific Blue™ isotype control (open histogram).
APC anti-human Ki-67
PHA-activated human peripheral blood lymphocytes (3 days) were fixed and permeabilized with 70% ethanol, and then stained with Ki-67 APC (filled histogram) or mouse IgG1, κ APC isotype control (open histogram).
3-day PHA-stimulated human peripheral blood lymphocytes were fixed and permeabilized with BioLegend FOXP3 buffer set, then stained with Ki-67 APC (filled histogram) or mouse IgG1, κ APC isotype control (open histogram).
