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AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
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AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
AlphaLISA Surefire Ultra Total Protein
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Sample Volume | 30 µL |
Product variants
Unit Size: 500 assay points
Part #:
ALSU-TCDK4-A500
Unit Size: 10,000 assay points
Part #:
ALSU-TCDK4-A10K
Unit Size: 50,000 assay points
Part #:
ALSU-TCDK4-A50K
Unit Size: 100 assay points
Part #:
ALSU-TCDK4-A-HV
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
Product information
Overview
Cyclin-dependent kinase 4 (CDK4) is a serine/threonine kinase that plays a pivotal role in cell cycle regulation, particularly in the transition from the G1 to S phase. CDK4 forms a complex with D-type cyclins to phosphorylate the retinoblastoma protein (Rb), leading to the release of E2F transcription factors and progression through the cell cycle. Dysregulation of CDK4 activity is frequently observed in various cancers, making it a critical target for therapeutic intervention. Inhibitors of CDK4, often in combination with CDK6 inhibitors, are used in the treatment of hormone receptor-positive breast cancer and are under investigation for other malignancies.
The AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit is a sandwich immunoassay for the quantitative detection of total CDK4 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.
In the AlphaLISA SureFire Ultra assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of protein present in the sample.
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 validation
PROTAC mediated degradation of CDK4
MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. Cells were treated with increasing concentrations of BSJ-03-204 PROTAC for 24 hours.
After treatment, the cells were lysed with 250 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK4 and Cyclin D1 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,600 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, BSJ-02-204 treatment induced a dose-dependent decrease in the levels of CDK4.
Assay versatility
Versatility of Total CDK4 assay in various cell lines
Adherent cells were seeded at 40,000 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 200,000 cells/well in a 96-well culture plate and lysed with 200 µL of Lysis Buffer.
CDK4 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 cells (adherent) or 10,000 cells (suspension)) 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.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Ultra
|
| Detection Modality |
Alpha
|
| Host Species |
Human
|
| Lysis Buffer Compatibility |
Lysis Buffer
|
| Molecular Modification |
Total
|
| Product Group |
Kit
|
| Sample Volume |
30 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
CDK4
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
100 assay points
|
Video gallery
AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Human Total CDK4 Detection Kit, 100 Assay Points
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Technical Note
PROTAC degraders targeting CDK4/CDK6 for the study of cell cycle regulation in oncology
The cell cycle is tightly regulated by key proteins like Cyclin D1, which forms complexes to initiate progression through phases...
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