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| Product Name | Ki-67 Proliferation Marker Immunoassay Kit, Colorimetric, Cell Lysate, Quantitative |
| Catalog No. | TMTR-HMM-0086 |
| Description | Quantitative colorimetric immunoassay kit for measuring Ki-67 (MKI67) protein, the gold-standard cellular proliferation marker, in cell and tissue lysates. Ki-67 is a nuclear protein expressed exclusively in actively cycling cells (G1, S, G2, and M phases) and absent in quiescent cells (G0 phase). The Ki-67 labeling index (percentage of Ki-67 positive cells by IHC) is a key prognostic and predictive biomarker in breast cancer, neuroendocrine tumors, prostate cancer, lymphoma, and many other malignancies — high Ki-67 indicates aggressive disease, higher grade, and greater benefit from chemotherapy. This ELISA-based format provides an objective, quantitative measurement alternative to subjective IHC scoring, enabling precise assessment of proliferation rates in cancer cell lines, tumor lysates, and drug response studies (anti-mitotics, CDK4/6 inhibitors). |
| Intended Use | Quantitative measurement of Ki-67 protein as a proliferation index in: cancer cell lines (HeLa, MCF-7, MDA-MB-231, HCT116, A549, Jurkat); tumor tissue lysates (breast, colon, lung, prostate, lymphoma); xenograft models; cell cycle synchronization studies; anti-proliferative drug screening (CDK4/6 inhibitors, chemotherapeutics); and growth factor/serum stimulation experiments. |
| Principle / Technology | Indirect immunoassay: cell/tissue lysates are coated onto the plate (or a capture antibody can be used — in this format, Ki-67 is captured from lysate onto a pre-coated anti-Ki-67 antibody plate). A biotinylated anti-Ki-67 detection antibody recognizing a distinct epitope binds the captured Ki-67. Streptavidin-HRP and TMB produce colorimetric signal. Quantitation against a Ki-67 peptide standard (containing both antibody epitopes) or recombinant Ki-67 fragment standard. |
| Detection Method | Lyse cells in RIPA buffer with protease/phosphatase inhibitors; centrifuge; dilute to 0.5-2 mg/mL; add 100 uL to wells; incubate 2 h RT; wash; detection antibody 1 h; wash; streptavidin-HRP 30 min; wash; TMB; stop; read A450; normalize Ki-67 signal to total protein or to a housekeeping protein control. |
| Sample Type | Cell lysates: 5-50 ug protein/well (proliferating cells yield strong signal; quiescent/serum-starved cells yield minimal signal); tumor tissue lysates: 20-100 ug/well; Ki-67 signal highly dependent on proliferation rate — serum-starved cells show 5-20x lower signal than exponentially growing cells. |
| Performance Range / Specifications | Standard curve: 0.31-20 U/mL (relative units calibrated against proliferating HeLa cell standard); LOD: <0.2 U/mL; linear: 0.62-20 U/mL R2 >0.99; proliferating HeLa: 8-15 U/mg protein; serum-starved HeLa: 0.5-2 U/mg. |
| Sensitivity / LOD | LOD <0.2 U/mL; Ki-67 detection in as few as 2,000 proliferating cells; sensitive to proliferation changes induced by 24 h serum starvation or CDK4/6 inhibitor treatment. |
| Specificity | Specific for human Ki-67 (MKI67 gene product, ~359 kDa and ~320 kDa isoforms); does not cross-react with PCNA, MCM2, phospho-histone H3 (Ser10), or other proliferation markers. |
| Reaction Conditions / Protocol | Capture 2 h RT; detection 1 h RT; HRP 30 min; TMB 15-30 min; total ~4-5 h. |
| Components / Formulation | Anti-Ki-67 Antibody Coated 96-Well Plate, Ki-67 Standard (lyophilized, HeLa lysate-derived or recombinant, calibrated in U/mL), Biotinylated Anti-Ki-67 Detection Antibody (100x), Streptavidin-HRP (100x), Sample Diluent, Standard Diluent, Wash Buffer (20x), TMB, Stop Solution, Plate Sealers, Protocol. |
| Storage Conditions | Plate and buffers at 2-8 C; Standard, antibodies, HRP at -20 C; protect from light. |
| Shelf Life | 12 months. |
| Package Specifications | 96 determinations. |
| Product Form | Pre-coated plate; liquid reagents; lyophilized standard. |
| Quality Control | Standard R2 >0.99; LOD <0.2 U/mL; intra-CV <8%; inter-CV <12%; proliferation-dependent signal: ratio of proliferating to quiescent HeLa signal >5:1; PCNA non-cross-reactivity confirmed. |
| Key Features | Quantitative Ki-67 immunoassay; objective alternative to IHC scoring; detects proliferation changes; HeLa standard for normalization; 96-well format; colorimetric detection. |
| Purity | Antibodies >95%; standard calibrated and validated; analytical grade reagents. |
| Concentration | Standard calibrated in relative units (U/mL); antibodies 100x concentrate. |
| Activity / Unit Definition | Anti-Ki-67 antibodies validated for ELISA and IHC applications. |
| Molecular Weight | Ki-67: ~359 kDa (major isoform), ~320 kDa (minor isoform); contains 16 tandem repeat elements (Ki-67 repeats) in the central domain. |
| Source / Origin | Recombinant monoclonal antibodies (animal-free production); standard from HeLa cell lysate or recombinant Ki-67 fragment; HRP plant-derived. |
| pH Range / Optimal pH | pH 7.2-7.4 optimal. |
| Shipping Conditions | Cold pack; antibodies and standard on dry ice. |
| Expiration Date / Stability | 12 months; reconstituted standard stable 1 week at 2-8 C or 3 months at -80 C; aliquot to avoid repeated freeze-thaw. |
| Regulatory / Compliance | For research use only; not for clinical diagnostic use. Not for Ki-67 labeling index determination in clinical breast cancer management. |
| Compatibility | RIPA lysis buffer; SDS <0.05% final; DTT/2-ME up to 1 mM; protease/phosphatase inhibitors essential; normalize to total protein (BCA) and report as U/mg protein. |
| Recommended Buffer System | PBS pH 7.4, 1% BSA, 0.05% Tween-20. |
| Application Notes / Precautions | Ki-67 is rapidly degraded at the end of M phase (proteasome-mediated); use fresh lysates and protease inhibitors. Proliferation conditions dramatically affect signal: include positive (exponentially growing cells) and negative (serum-starved or confluent) controls. For drug studies: treat cells with anti-proliferative agents for 24-72 h; normalize signal to total protein or cell count. Ki-67 peptide standards calibrated in relative units — this is a semi-quantitative assay for comparing relative proliferation between samples, not an absolute molar quantitation. For tissue: the Ki-67 ELISA signal correlates with IHC labeling index (R ~0.7-0.9 in published studies) but should be validated for each tumor type. |
| Batch-to-Batch Consistency | Standard R2 >0.99; LOD <0.2 U/mL; proliferating/quiescent ratio >5:1; inter-lot CV <15%. |
For research use only, not for clinical use.
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