Ki-67 Monoclonal Antibody (20Raj1), PerCP-eFluor™ 710, eBioscience™

Product Specific Information

Description: The monoclonal antibody 20Raj1 recognizes the human Ki-67 protein. Two isoforms of Ki-67 exist, a 345 and 395 kDa form that are expressed in dividing cells. Ki-67 is expressed in all cell types and is detectable during active phases of the cell cycle (G1, S, G2, and mitosis) but is absent from resting cells (G0). During interphase, Ki-67 expression is localized to the nucleus but redistributes to the chromosomes during mitosis and has specifically been found to associate with heterochromatin-bound proteins such as chromobox protein homolog 3 (CBX3). In studies of tumor cells, Ki-67 expression has been used as a marker for determining the fraction of proliferating cells within a given population of tumor cells.

This monoclonal antibody 20Raj1 recognizes canine Ki-67.

Applications Reported: This 20Raj1 antibody has been reported for use in intracellular staining using the Foxp3 Staining Buffers followed by flow cytometric analysis.

Applications Tested: This 20Raj1 antibody has been pre-titrated and tested by intracellular staining using Foxp3/Transcription Factor Buffers Set (Product # 00-5523-00) and protocol of normal human peripheral blood cells. Please see BestProtocols® Section (Staining intracellular Antigens for Flow Cytometry) for staining protocol (refer to Protocol B: One-step protocol for intracellular (nuclear) proteins. This can be used at 5 µL (0.03 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

Ki-67 is a nuclear protein that is expressed during various stages in the cell cycle, particularly during late G1, S, G2, and M phases. The protein has a forkhead associated domain (FHA) through which it associates with euchromatin at the perichromosomal layer, the centromeric heterochromatin, and the nucleolus. Ki-67 is shown to have a cell cycle dependent topographical distribution with perinucleolar expression at G1, expression in the nuclear matrix at G2, and expression on the chromosomes during M phase. Ki-67 is commonly used as a proliferation marker because it is not detected in G0 cells, but increases steadily from G1 through mitosis. Ki-67 antibodies are useful in establishing the cell growing fraction in neoplasms. In neoplastic tissues, the prognostic value is comparable to the tritiated thymidine-labelling index. The correlation between low Ki-67 index and histologically low-grade tumors is strong. Ki-67 is routinely used as a neuronal marker of cell cycling and proliferation.