Immunofluorescent analysis of HeLa cells costained with chicken polyclonal antibody to Ki-67 CPCA-Ki67, dilution 1:2,000 in red and monoclonal antibody to β-tubulin MCA-1B12 , dilution 1:5,000 in green. Ki-67 is predominantly localized in nucleoli of cells in interphase, while in mitotic cells Ki-67 is associated with condensed chromosomes. Ki-67 is not expressed in cells in quiescent G0 state. The blue is DAPI staining of nuclear DNA.
|Western blots analysis of equal amounts of cell lysates using chicken polyclonal antibody to Ki-67 CPCA-Ki67, dilution 1: 5,000 in green:  protein standard (red),  HeLa cells,  HEK293 cells. Strong double bands above 250 kDa mark correspond to two major isoforms of molecular weight 345 and 395 kDa of Ki-67 protein. Smaller fragments of these isoforms are also detected on the blot.
||Chicken polyclonal antibody to Ki-67
||345 and 395kDa
||Mixture of recombinant constructs of human Ki-67 :1-300 and 1,111-1,490 (P46013) amino acids expressed in and purified from E. coli.
||Supplied as an aliquot of IgY preparation.
||Western blot, ICC/IF, IHC
|Suggestions for use
||WB: 1:2,000-5,000. IF/IHC: 1:1,000-5,000.
||Store at 4°C for short term and -20°C for long term. Avoid repeated freeze / thaw cycles.
The Ki-67 protein was first discovered in an attempt to generate cancer specific monoclonal antibodies. Mice were injected with nuclear preparations from Hodgkin’s lymphoma cells. One hybridoma produced an antibody which strongly stained the nucleoli of dividing but not quiescent cells. The antibody was named Ki-67; the name was derived of Kiel, Germany which was the location of the work, and the number 67 was the well number in which hybridoma was first identified (1). The Ki-67 antibody recognizes a very large protein with two isoforms of 345 kDa and 395 kDa. Western blots often show a ladder of smaller fragments of these isoforms since the proteins are rather unstable, consistent with their short in vivo half-life of about 1 hour.
The proteins were found to be heavily expressed in proliferating cells, but to be absent in quiescent cells (2). Ki-67 protein is frequently used as an indicator of cell proliferation and its level of expression is one of the most reliable biomarkers of proliferative status of cancer cells. Much research shows a correlation between Ki-67 protein level and prognosis in cancer patients, when high Ki-67 levels being associated with poorer outcomes (3-5). The original Ki-67 antibody and several others have become so widely used that a search for “Ki-67” in PubMed early in 2017 produced almost 23,000 results. Recent studies show that Ki-67 functions as a “biological surfactant”, which is essential for the separation of condensed chromosomal DNA into the two daughter cells during cell division (6). This presumably explains the highly basic nature of Ki-67, allowing a charge-based interaction with nucleic acids, and the lack of this protein in non-dividing cells.
EnCor chicken polyclonal antibody to Ki-67 (CPCA-Ki-67) raised against a mixture of two recombinant constructs bases on the human sequence. One contains amino acids 1-300 and includes the forkhead associated domain and a region of low sequence complexity. The other includes amino acids 1,111-1,490, corresponding to the second, third and fourth of the sixteen 110 amino acid Ki-67 sequence repeats of human Ki-67 isotype 1, as laid out in the Uniprot entry P46013. The Ki-67 protein sequence is quite variable across species, and an antibody to the human protein often fails to recognize the rodent homologue and vice versa. As a result, this antibody is not recommended for studies of rodents.
1. Gerdes J, Schwab U, Lemke H. and Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer 31:13-20 (1983).
2. Kill I R, Faragher RGA, Lawrence K. and Shall S. The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture. J. Cell Sci. 107: 571-579 (1994).
3. Yerushalmi R, et al. Ki67 in breast cancer: Prognostic and predictive potential. Lancet Oncol. 11: 174–183 (2010).
4. Josefsson A, et al. Low endoglin vascular density and Ki67 index in Gleason score 6 tumours may identify prostate cancer patients suitable for surveillance. Scand J Urol Nephrol. 46: 247–257 (2012).
5. Ishihara M, et al. Retrospective analysis of risk factors for central nervous system metastases in operable breast cancer: effects of biologic subtype and Ki67 overexpression on survival. Oncology. 84: 135–140 (2013).
6. Cuylen S, et al. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.Nature. 535: 308-12 (2016).
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