Hartland Jackson 952x200 CMS (EVENT USE ONLY)

Ki67 target tips

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

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Overview

​​Sample fixation

Antigen retrieval and permeabilization

Recommended controls

​Sample fixation

Permeabilization

Recommended controls


​​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 the late G1, S, G2, and M phases of the cell cycle, however, the antigen cannot be detected (at protein level) in cells in the G0 phase 4.    
  • Ki67 is present at the highest level in the 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)

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IHC tips

Ki67 is located in the nucleus, so you need to permeabilize cells for successful immunostaining. We recommend following the tips below for IHC.


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

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

Over-fixation can mask the epitope; antigen retrieval can help overcome this masking, but if the tissue has been fixed for too long (i.e. over a weekend), there may be no signal even after antigen retrieval.

  • We recommend fixation with 4% PFA or 10% formalin.

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Antigen retrieval and permabilization


Antigen retrieval: Heat in citrate buffer, pH 6, 20 – 30 mins.

If the protein is fixed in PFA, then enzymatic antigen retrieval with proteinase K or trypsin is necessary. 


Permeabilization: ​Incubate in 0.2 % Triton in PBS for 10 minutes.

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Recommended controls


Positive: Human tonsil tissue; mouse tumour tissue; mouse embryonic skin tissue; rat oesophagus, small intestine and liver tissue.

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ICC tips

Ki67 is located in the nucleus, so you need to permeabilize cells for successful immunostaining. We recommend following the tips below for IHC.

Sample fixation

We recommend fixing cells in 4% PFA for 20 minutes at room temperature.

Be sure not to over-fix your samples, as this will reduce the signal.

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Permeabilization

Incubate cells with 0.1 % Triton-X for 5 mins.

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Recommended Controls

Positive: HeLa cells; HAP1; rat cardiomyocytes

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Download these tips as a PDF: 

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)