Description
Overview
Cyclin-dependent kinase 6 (CDK6) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells. Mitogenic signals, such as growth factors, trigger cells to enter the G1 phase of the cell cycle by inducing Cyclin D synthesis. Cyclin D then interacts with CDK4 and CDK6 to form active complexes. Both activated kinases are then able to mono-phosphorylate the tumor suppressor RB (protein of retinoblastoma), which still binds to transcription factor E2F, but some genes can be transcribed, such as Cyclin E. CDK6 is overexpressed in many cancers such as lymphoma, leukemia, glioblastoma, bladder, pancreas, and prostate.
The AlphaLISA
SureFire
Human Total CDK6 Detection Kit is a sandwich immunoassay for the quantitative detection of total CDK6 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
Alpha
SureFire
kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
How it works
Total-AlphaLISA
SureFire Ultra
assay principle
The Total-AlphaLISA
SureFire Ultra
assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA
SureFire Ultra
assay uses two antibodies which recognize two different distal epitopes on the targeted 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 targeted 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 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 permits the cells 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 AlphaLISA
SureFire Ultra
quality.
Assay versatility
CDK6 expression in various cell lines
Adherent cells were seeded at 50,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer.
Suspension cells were seeded at 50,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 100 µL of Lysis Buffer. CDK6 Total levels were evaluated using the AlphaLISA
SureFire Ultra
assay. For the detection step, 10 µL of cell lysate (approximately 5,000 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.
CDK6 is expressed across a wide range of human cell types.
