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AlphaLISA SureFire Biotin-Free Human and Mouse Total CDK2 Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Biotin-Free Human and Mouse Total CDK2 Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total CDK2 assay is a sandwich immunoassay for quantitative detection of total CDK2 in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Protocol Time | 2h at RT |
| Sample Volume | 10 µL |
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total CDK2 assay is a sandwich immunoassay for quantitative detection of total CDK2 in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 Assay Points
Part #:
ASBF-TCDK2-A-HV
Unit Size: 500 Assay Points
Part #:
ASBF-TCDK2-A500
Unit Size: 10,000 Assay Points
Part #:
ASBF-TCDK2-A10K
Unit Size: 50,000 Assay Points
Part #:
ASBF-TCDK2-A50K
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 Biotin-Free Human and Mouse Total CDK2 Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
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Product information
Overview
Cyclin-Dependent Kinase 2 (CDK2) is a serine/threonine kinase that plays essential roles in cell cycle progression, particularly at the G1/S transition and during S phase. CDK2 activity is regulated by association with cyclin E (during late G1) and cyclin A (during S phase and G2), as well as by activating phosphorylation at Thr160 by CDK-activating kinase (CAK) and inhibitory phosphorylation at Thr14/Tyr15 by WEE1 and MYT1. CDK2/cyclin E complexes phosphorylate the retinoblastoma protein (Rb), releasing E2F transcription factors to drive S phase entry, while CDK2/cyclin A complexes regulate DNA replication and prevent re-replication. CDK2 is frequently dysregulated in cancer through cyclin E amplification, loss of CDK inhibitors such as p27 (CDKN1B) and p21 (CDKN1A), or Rb pathway inactivation, leading to aberrant S phase entry and genomic instability. CDK2 inhibitors are under active clinical investigation, with selective CDK2 inhibitors showing particular promise in cyclin E1-amplified cancers and as combination partners with CDK4/6 inhibitors to overcome resistance mechanisms in hormone receptor-positive breast cancer.
The AlphaLISA SureFire Biotin-Free Human and Mouse Total CDK2 Detection Kit is a sandwich immunoassay for the quantitative detection of total CDK2 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 Biotin Free kits are compatible with:
- Cell and tissue lysates
- Antibody modulators
- Biotherapeutic antibodies
- Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
- Cellular kinase assays
- Receptor activation studies
- High-throughput screening for preclinical studies
How it works
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Biotin Free 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 a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. 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 Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Biotin Free 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 quality.
Assay validation
Induction of CDK2 in MCF7 cells treated with Neuregulin-1
MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with 5µM LY294002 (PI3K inhibitor) for 3 hours then with increasing concentrations of Neuregulin-1 (NRG-1) for 24 hours in serum free media.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK2 Phospho (Thr160) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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, NRG-1 treatment resulted in a dose-dependent increase of Phospho (Thr160) and Total CDK2 levels.
CDK2 degradation in cells treated with CPS2
Ramos cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 and treated with increasing concentrations of CPS2 for 24 hours at 37°C, 5% CO2.
After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK2 Total and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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 CPS2, a CDK2 PROTAC, resulted in a decrease of CDK2 levels while Total ERK levels remained unchanged.
Inhibition of CDK2 phosphorylation in cells treated with palbociclib
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of palbociclib for 24 hours at 37°C, 5% CO2.
After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Cell lysates were further diluted in Lysis Buffer and then CDK2 Phospho (Thr160) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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, palbociclib treatment resulted in a dose-dependent decrease of CDK2 Phospho (Thr160) with a modest decrease of Total levels.
CDK2 phosphorylation decreases in cells treated with CDK4/6 PROTACs
MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium incubated overnight at 37°C, 5% CO2.Cells were treated with increasing concentrations of BSJ-03-123 or BSJ-03-204 for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK2 Phospho (Thr160) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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 CDK4/6 PROTACs resulted in a dose-dependent decrease of CDK2 Phospho (Thr160) while Total levels remained unchanged.
Assay sensitivity
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from HeLa cells seeded in a T175 flask cultured to confluence and lysed in 5 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and CDK2 Phospho (Thr160) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free. 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 per datapoint is indicated. The dotted line represents assay background. The assay can detect CDK2 expression in less than 100 cells.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Biotin-Free
|
| Detection Modality |
Alpha
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Sample Volume |
10 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
CDK2
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
50,000 Assay Points
|
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