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AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points

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AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points
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AlphaLISA SureFire Biotin Free Total Kit Schematic
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The AlphaLISA™ SureFire® Biotin-Free Human Total CDK1 assay is a sandwich immunoassay for quantitative detection of total CDK1 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 Total CDK1 assay is a sandwich immunoassay for quantitative detection of total CDK1 in cellular lysates using Alpha Technology.

View product information
Product Variants
Unit Size: 100 assay points
Part #:
ASBF-TCDK1-A-HV
Unit Size: 500 assay points
Part #:
ASBF-TCDK1-A500
Unit Size: 10,000 assay points
Part #:
ASBF-TCDK1-A10K
Unit Size: 50,000 assay points
Part #:
ASBF-TCDK1-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-Biotin-Free-Total-Primary
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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AlphaLISA-SureFire-Biotin-Free-Total-Primary
AlphaLISA SureFire Biotin Free Total Kit Schematic
AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points
AlphaLISA-SureFire-Biotin-Free-Total-Primary
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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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 Total CDK1 Detection Kit is a sandwich immunoassay for the quantitative detection of total 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
50,000 assay points

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AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points
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AlphaLISA SureFire Biotin-Free Human Total CDK1 Detection Kit, 50,000 Assay Points
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How it works

Total-AlphaLISA SureFire Biotin Free assay principle

The Total-AlphaLISA Surefire Biotin Free assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA Surefire 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. 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 Biotin Free assay principle

 

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 permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2 plates assay protocol alphalisa surefire ultra biotin free total assay
Total-AlphaLISA SureFire 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.

1 plate assay protocol AlphaLISA Surefire Ultra Total assay

Assay validation

Validation of CDK1 Total in Hydroxyurea treated cells

MOLT-4 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium 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 Total and Phospho (Thr14) 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 Total CDK1.

Pharmacological Validation of CDK1 Total
Nocodazole induces changes in the levels of CDK1 Total

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, 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.

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

Pharmacological Validation (inhibitor) of CDK1 Total

Resources

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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments

The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay

Several biological processes are regulated by...

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Alpha SureFire Ultra no-wash immunoassay catalog

Discover Alpha SureFire® Ultra™ assays, the no-wash cellular kinase assays leveraging Revvity's exclusive bead-based technology...

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Technical Note
Introducing AlphaLISA SureFire® Biotin Free: A Versatile Immunoassay Platform for Biotin-Rich Samples.

Featuring homogeneous detection of cell-based proteins, this note shows you how to utilize an innovative system optimized for...

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Species compatibility for HTRF, AlphaLISA SureFire Ultra and Alpha SureFire Ultra Multiplex assays

This document includes detailed tables listing HTRF™, AlphaLISA™ SureFire® Ultra™, and Alpha SureFire® Ultra™ Multiplex assays...

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