Figure 1. Ki-67 protein is detected at varying levels from multiple cell lines using the PathScan® Total Ki-67 Sandwich ELISA Kit #14507. The absorbance readings at 450 nm are shown in the top figure, while corresponding western blots using Ki-67 (D3B5) Rabbit mAb #9129 are shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from HeLa cells and the absorbance at 450 nm as detected by the PathScan® Total Ki-67 Sandwich ELISA Kit #14507 is shown. HeLa cells (85% confluence) were harvested and then lysed.
|Product Includes||Volume||Solution Color|
|Ki-67 Rabbit mAb Coated Microwells||96 tests|
|Ki-67 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Total Ki-67 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Ki-67. A Ki-67 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, Ki-67 protein is captured by the coated antibody. Following extensive washing, a Ki-67 Mouse Detection mAb is added to detect the captured Ki-67 protein. An HRP-linked, anti-mouse antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total Ki-67.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total Ki-67 Sandwich ELISA Kit detects endogenous levels of Ki-67 protein as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Ki-67, named after the location where it was discovered (Kiel University, Germany), is a nuclear nonhistone protein (1) that is universally expressed among proliferating cells and absent in quiescent cells (2). Ki-67 detects proliferating cells in G1, S, G2, and mitosis, but not in the G0 resting phase. Research studies have shown that high levels of Ki-67 are associated with poorer breast cancer survival (3). Research studies have explored the use of Ki-67, along with other markers, as potential prognostic or predictive markers in breast cancer and other malignant diseases (4).
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PathScan is a trademark of Cell Signaling Technology, Inc.