Ki-67 (MKI67) is a nuclear protein that serves as a proliferation marker expressed during all active phases of the cell cycle (G1, S, G2, and M) but absent in quiescent cells (G0). Ki-67 localizes to the nucleolus during interphase and associates with condensed chromosomes during mitosis, playing roles in ribosomal RNA synthesis and chromatin organization. The precise molecular function of Ki-67 remains incompletely understood, but it appears essential for maintaining chromosome structure during mitosis. Ki-67 expression is widely used as a prognostic and predictive biomarker in cancer, with high Ki-67 index indicating aggressive tumor behavior and poor prognosis in breast cancer, lymphomas, and neuroendocrine tumors. Ki-67 immunohistochemistry guides treatment decisions, particularly in determining the need for chemotherapy in hormone receptor-positive breast cancer.
The AlphaLISA SureFire Ultra Human Total Ki-67 Detection Kit is a sandwich immunoassay for the quantitative detection of total Ki-67 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Decrease of Ki-67 Total levels in palbociclib treated cells
MCF7 cells were seeded in a 96-well plate at 40,000 in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of palbociclib for 24 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and GAPDH Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, treatment with palbociclib resulted in a dose-dependent decrease of Ki-67 levels, while GAPDH Total levels remained unchanged.
A549 cells were seeded in a 96-well plate at 20,000 cells/well in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of palbociclib for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of diluted cell lysate (approximately 800 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, treatment with palbociclib resulted in a dose-dependent decrease of Ki-67 levels, while ERK Total levels remained unchanged.
Decrease of Ki-67 levels in Dinaciclib treated cells
T-47D cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of Dinaciclib for 24 hours.
After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 cells for Ki-67 and 240 cells for Cofilin) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Dinaciclib triggered a dose-dependent decrease in Ki-67 levels, while Cofilin Total remained unchanged.
CDK6 PROTAC-mediated downregulation of Ki-67 expression in A549 cells
A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. Cells were treated with 10 µM of CDK6 PROTAC, BSJ-03-123 for 24 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67, CDK6 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, degradation of CDK6 lead to Ki-67 downregulation in A549 cells while ERK Total levels remained unchanged.
CDK4/6 PROTAC-mediated downregulation of Ki-67 expression in A549 cells
A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of CDK 4/6 PROTAC BSJ-03-204 for 24 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67, CDK6 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, degradation of CDK4 and CDK6 lead to Ki-67 downregulation in A549 cells while no significant changes were observed in ERK Total levels.
Knockout validation of Ki-67 Total assay
Ki-67 Total protein levels were assessed in HeLa wild type (WT) and Ki-67 knockout (KO, Abcam, ab255407) cell lines cultured to confluency in T175 flasks. Each flask was lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and evaluated for Ki-67 expression using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Ki-67 expression was only detected in WT cells, demonstrating assay specificity. Dotted line represents assay background.
Ki-67 expression in various cell lines
Adherent cells were grown to confluency in a T175 flask and lysed with Lysis Buffer at a density of 0.125 x 106 cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at a density of 0.4 x 106 cells/mL.
Ki-67 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (1,250 adherent and 4,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Ki-67 expression was detected in a wide range of human cell lines.
Ki-67 assay sensitivity - cell lysate
Cell lysate was prepared from A459 cells cultured to confluence in T175 flasks in complete medium. Cells were lysed in 8 mL of Lysis Buffer.
Lysate was serially diluted in Lysis Buffer and Ki-67 levels were assayed using the AlphaLISA SureFire Ultra kit. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Approximate number of cells/datapoint is indicated. The dotted line represents assay background. The assay can detect Ki-67 expression in less than 200 cells.
