Ki67

Ki67 is a nuclear protein encoded by the MKI67 (marker of proliferation ki-67) gene. Ki67 play key roles in cell cycle, cell proliferation and ribosomal RNA transcription.

Overview

​​Protein function, expression, and isoforms

Protein function 

  • ​Ki67 is required to maintain individual mitotic chromosomes dispersed in the cytoplasm.1
  • It associates with the surface of the mitotic chromosome, the perichromosomal layer, and covers a substantial fraction of the chromosome surface. 1
  • Ki67 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.1
  • It binds DNA with a preference for supercoiled DNA and AT-rich DNA.2
  • It may play a role in chromatin organization.3

​​Expression

  • 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).4     
  • Ki67 is present at highest level in G2 phase and during mitosis (at protein level). 
  • In interphase, Ki67 forms fiber-like structures in fibrillarin-deficient regions surrounding nucleoli. 5, 6

​​Isoforms

  • ​Human: Isoform 1: 319 kD (predicted)
  • Human: Isoform 2: 359 kD (predicted)
  • Mouse: Isoform 1: 325 kD (predicted)
  • Mouse: Isoform 2: 351 kD (predicted)
  • Rat: Isoform 1: 343 kD (predicted)

Applications

Ki67 can be a challenging target to work with in some applications.

Immunohistochemistry

Expression of Ki67 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).6



Sample fixationThe ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18–24h is suitable for most samples.

Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle.

Over-fixation can mask the epitope; antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.
Maximizing signalAntigen retrieval: Heat in citrate buffer pH 6 for 20-30 minutes or enzymatic (trypsin, proteinase K). (Necessary if fixed in PFA)

Permeabilize the tissues: 0.2% Triton in PBS for 10 minutes
ControlsPositive: 
Human tonsil tissue
Mouse tumour tissue
Mouse embryonic skin tissue
Rat oesophagus, small intestine and liver tissue

​​Immunocytochemistry/Immunofluorescence

Ki67 locates in chromosomes and nucleus. Therefore, 4% PFA fixation is recommended. And permeabilizing the cells (0.1% TritonX-100 in PBS for 5 minutes) in ICC assays is essential.



Sample fixation For nuclear proteins, fix cells in 4% PFA (20 minutes, room temperature) is recommended.

Do not over-fix your samples, as this will reduce signal.
PermeabilizationIt is recommended to incubate cells with 0.1% Triton-X for 5 min to detect nuclear antigen.
ControlsPositive: 
HeLa and HAP1 cells
Rat cardiomyocytes

Find full information on working with Ki67: 

References

  1. Cuylen, S., Blaukopf, C., Politi, A.Z., et al. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature. 535, 308-12 (206)
  2. MacCallum, D.E., Hall, P.A. The biochemical characterization of the DNA binding activity of pKi67. J Pathol. 19, 286-98 (2000)
  3. Booth, D.G., Takagi, M., Sanchez-Pulido, L., et al. Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery. Elife. 27, e01641 (2014)
  4. Gerdes, J., Lemke, H., Baisch, H., Wacker, H.H., Schwab, U., Stein, H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 133, 1710-5 (1984)
  5. Verheijen, R., Kuijpers, H.J., Schlingemann, R.O., et al. Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase. J Cell Sci. 92, 123-30 (1989)
  6. Kill, I.R. Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component. J Cell Sci. 109, 1253-63 (1996)