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HTRF Human and Mouse Phospho-CDK1 Thr14 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated CDK1 (Cyclin-Dependent Kinase 1) at Thr14, which is an inhibitory phospho-site essential for maintaining genome integrity and preventing DNA damage during the G2-M phase transition.

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  • No-wash No-wash
  • High sensitivity High sensitivity
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption

This HTRF kit enables the cell-based quantitative detection of phosphorylated CDK1 (Cyclin-Dependent Kinase 1) at Thr14, which is an inhibitory phospho-site essential for maintaining genome integrity and preventing DNA damage during the G2-M phase transition.

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Overview

This HTRF cell-based assay conveniently and accurately detects phosphorylated CDK1 at Thr14. ​​​​​​​

CDK1 (Cyclin-Dependent Kinase 1) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (also including CDK1, CDK4, and CDK6). CDK1 is a catalytic subunit of a protein kinase called the M-phase promoting factor that induces entry into mitosis. CDK1 promotes G2-M transition, and regulates G1 progress and G1-S transition via association with multiple interphase cyclins (Cyclin A, Cyclin B).  Phosphorylation at Thr14 and Tyr15, resulting in inhibition of CDK1, can be carried out by Wee1 and Myt1 protein kinases . The cdc25 phosphatase may be responsible for the removal of phosphates at Thr14 and Tyr15 and subsequent activation of CDK1.

CDK1 inhibitory phosphorylation at Thr14 is essential for maintaining genome integrity and preventing DNA damage during the S phase. The Wee1/Cdc25A axis is therefore an attractive target for cancer therapy and may represent a unique approach to sensitize cancer cells with hyperactive CDK1.


Benefits

  • SPECIFICITY
  • PRECISION

Phospho-CDK1 (Thr14) assay principle

The Phospho-CDK1 (Thr14) assay measures CDK1 when phosphorylated at Thr14. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Principle of the HTRF Phospho CDK1 (Thr14)assay

Phospho-CDK1 (Thr14) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-CDK1 (Thr14) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Phospho CDK1 (Thr14) assay

Phospho-CDK1 (Thr14) one-plate assay protocol

Detection of Phosphorylated CDK1 (Thr14) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

One-plate protocol of the HTRF Phospho CDK1 (Thr14) assay

Phospho-CDK1 (Thr14) modulation using Hydroxyurea

HeLa cells were cultured in a 96-well plate (50,000 cells/well) for 6h, and then treated overnight with increasing concentration of Hydroxyurea (inducer of single strand breaks). After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-CDK1 (Thr14) or Total CDK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, hydroxyurea triggered a dose-dependent increase in phosphorylated CDK1 at Thr14, while the expression level of the protein was not modulated by the treatment.

Phospho CDK1 (Thr14) modulation using Hydroxyurea

Phospho-CDK1 (Thr14) modulation using Wee1/Myt1 inhibitor

HeLa and MCF7 cells were cultured in a 96-well plate (50,000 cells/well) for 24h, and then treated for 2h with the Wee1/Myt1 kinase inhibitor PD0166285.

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-CDK1 (Thr14) or Total CDK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, the Wee1/Myt1 kinase inhibitor PD166285 decreased phosphorylated CDK1 at Thr14, while the expression level of the protein remained stable.

Inhibition of Phospho CDK1-T14 on HeLa and MCF7 cells
Inhibition of Phospho CDK1-T14 on HeLa and MCF7 cells

Validation of Phospho-CDK1 (Thr14) assay specificity by siRNA knockdown experiments

HeLa cells were plated in a 96-well plate (10,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for CDK1, CDK2, CDK3, CDK4, CDK5, or CDK6, as well as with a negative control siRNA. After a 48h incubation, the cells were lyzed. 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-CDK1 (Thr14) detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cell transfection with the CDK1 siRNA led to a 72% signal decrease compared to the cells transfected with the negative siRNA. On the contrary, the knockdown of CDK2, CDK3, CDK4, CDK5, or CDK6 did not induce any signal decrease, demonstrating that the HTRF Phospho-CDK1 (Thr14) assay is specific for CDK1 phosphorylation and does not cross-react with other cell cycle CDK family members.

Specificity of Phospho CDK1-T14 assay using SiRNA

Assessment of phospho CDK1 Thr14 levels in various cell lines

Adherent human & mouse cells HeLa, MCF7, and Neuro 2A,  or suspension, such as THP1 cells, were seeded at 50,000 cells / well in a 96-well microplate. After a 24H incubation, the cells were lyzed with supplemented lysis buffer, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho CDK1 (Thr14) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF phospho CDK1 (Thr14) assay efficiently detected phospho CDK1 (Thr14) in various cellular models expressing different levels of the protein.

Validation in various cell lines

HTRF phospho-CDK1 (Thr14) assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were  lyzed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-CDK1 (Thr14) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-CDK1 (Thr14) assay, 625 cells/well were enough to detect a significant signal, while 20,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-CDK1 (Thr14) assay was 32 times more sensitive than the Western Blot technique.

Comparison of WB with HTRF Phospho CDK1-T14 assay

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Oncology Guide

Cellular Signaling Processes Involved in Cancer Development and Progression - Guides

Manual CDK1 p-T14 Kit / 64CDK1T4PEG-64CDK1T4PEH

64CDK1T4PEG-64CDK1T4PEH - Product Insert

Certificate of Analysis CDK1 p-T14 Kit / 64CDK1T4PEG

64CDK1T4PEG Batch 01C - Quality Control Report

Certificate of Analysis CDK1 p-T14 Kit / 64CDK1T4PEG

64CDK1T4PEG Batch 01D - Quality Control Report

Certificate of Analysis CDK1 p-T14 Kit / 64CDK1T4PEG

64CDK1T4PEG Batch 01E - Quality Control Report

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