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AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points

AlphaLISA SureFire Ultra Phospho-Protein image
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AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
Video Play Button
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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The AlphaLISA™ SureFire® Biotin-Free Human Phospho-CDK1 (Thr14) assay is a sandwich immunoassay for quantitative detection of phospho-CDK1 (Thr14) in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Biotin-Free Human Phospho-CDK1 (Thr14) assay is a sandwich immunoassay for quantitative detection of phospho-CDK1 (Thr14) in cellular lysates using Alpha Technology.

View product information
Product Variants
Unit Size: 100 assay points
Part #:
ASBF-PCDK1-A-HV
Unit Size: 500 assay points
Part #:
ASBF-PCDK1-A500
Unit Size: 10,000 assay points
Part #:
ASBF-PCDK1-A10K
Unit Size: 50,000 assay points
Part #:
ASBF-PCDK1-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).
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AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
Video Play Button
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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Product information

Overview

Cyclin-dependent kinase 1 (CDK1) is a serine/threonine kinase critical for cell cycle regulation. It partners with cyclin B to control the G2/M transition, ensuring proper mitotic progression through phosphorylation of key substrates. CDK1 overexpression is common in cancers, such as breast and lung cancers, promoting uncontrolled proliferation. Dysregulated CDK1 activity is also linked to neurodegenerative diseases like Alzheimer's, where it contributes to neuronal cell death.

The AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-CDK1 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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Biotin-Free
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
CDK1
Target Class
Phosphoproteins
Target Species
Human
Technology
Alpha
Therapeutic Area
Neuroscience
Oncology
Unit Size
500 assay points

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AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points
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AlphaLISA SureFire Biotin-Free Human Phospho-CDK1 (Thr14) Detection Kit, 500 Assay Points
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How it works

Phospho-AlphaLISA SureFire Biotin Free assay principle

The Phospho-AlphaLISA SureFire Biotin Free assay measures a protein target when phosphorylated at a specific residue.

The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. In the presence of phosphorylated 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 phosphoprotein present in the sample.

assay principle Phospho AlphaLISA Surefire Ultra Biotin Free

 

Phospho-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 Phospho-AlphaLISA SureFire Biotin Free 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.

Phospho-AlphaLISA SureFire Ultra Biotin Free two-plate assay protocol
Phospho-AlphaLISA SureFire Biotin Free one-plate assay protocol

Detection of Phosphorylated 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.

Phospho-AlphaLISA SureFire Ultra Biotin Free one-plate assay protocol

Assay validation

Activation of CDK1 Phospho (Thr14) in Hydroxyurea treated cells

MOLT-4 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 and treated with increasing concentrations of Hydroxyurea for 18 hours at 37°C, 5% CO2.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK1 Phospho (Thr14) and CDK1 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysates were further diluted 1:4 in Lysis Buffer, 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, Hydroxyurea triggered a dose-dependent increase in the levels of Phospho CDK1 (Thr14) with no significant changes in CDK1 Total.

Activation of CDK1 Phospho (Thr14) in Hydroxyurea Treated Cells
Decrease of CDK1 Phospho (Thr14) in Nocodazole treated cells

Jurkat cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 and treated with increasing concentrations of Nocodazole for 18 hours at 37°C, 5% CO2.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK1 Phospho (Thr14) and CDK1 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:4 in Lysis Buffer, 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.

Nocodazole triggered a significant decrease in the levels of Phospho CDK1 (Thr14) with a modest decrease in Total CDK1 levels in Jurkat cells.

Decrease of CDK1 Phospho (Thr14) in Nocodazole Treated Cells

Resources

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