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HTRF Human and Mouse Phospho-CDK1 Tyr15 Detection Kit

HTRF Human and Mouse Phospho-CDK1 Tyr15 Detection Kit HTRF®

Name: HTRF Human and Mouse Phospho-CDK1 Tyr15 Detection Kit
Brand: Cisbio
SKU: 46242
Availability: InStock

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This HTRF kit enables the cell-based quantitative detection of phosphorylated CDK1 (Cyclin-Dependent Kinase 1) at Tyr 15, 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
High sensitivity
All inclusive kit
Low sample consumption

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Overview

This HTRF cell-based assay conveniently and accurately detects phosphorylated CDK1 at Tyr15.

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 Tyr15 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.

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Phospho-CDK1 (Tyr15) assay principle

The Phospho-CDK1 (Tyr15) assay measures CDK1 when phosphorylated at Tyr15. 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 (TYR15) assay

Phospho-CDK1 (Tyr15) 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 (Tyr15) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Phospho CDK1 (Tyr15) assay

Phospho-CDK1 (Tyr15) one-plate assay protocol

Detection of Phosphorylated CDK1 (Tyr15) 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 (Tyr15) assay

Phospho-CDK1 (Tyr15) 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 (Tyr15) 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 Tyr15, while the expression level of the protein was not modulated by the treatment.

Phospho CDK1 (Tyr15) modulation using Hydroxyurea

Phospho-CDK1 (Tyr15) modulation using Wee1/Myt1 inhibitor

HeLa cells were cultured in a 96-well plate (50,000 cells/well) for 24h and then treated for 2h with the Wee1 kinase inhibitors Adavosertib and 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 (Tyr15) or Total CDK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, both Wee1 kinase inhibitors triggered a dose-dependent decrease in phosphorylated CDK1 at Tyr15, while the expression level of the protein was not modulated by the treatments.

Inhibition of Phospho CDK1 (Tyr15) on HeLa cells

Validation of Phospho-CDK1 (Tyr15) assay specificity by siRNA knockdown experiments

HeLa cells were plated in a 96-well plate (5,000 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 and 16 µL of lysates were transferred into a 384-well low volume white microplate. 4 µL of the HTRF Phospho-CDK1 (Tyr15) detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cell transfection with the CDK1 siRNA led to a 68% 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 (Tyr15) assay is specific for CDK1 phosphorylation and does not cross-react with other cell cycle CDK family members.

Specificity of Phospho CDK1 (Tyr15) assay using SiRNA

Assessment of phospho CDK1 Tyr15 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 (Tyr15) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF phospho CDK1 (Tyr15) assay efficiently detected phospho CDK1 (Tyr15) in various cellular models expressing different levels of the protein.

Validation in various cell lines HTRF phospho CDK1Y15

HTRF phospho-CDK1 (Tyr15) 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 (Tyr15) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-CDK1 (Tyr15) assay, 1250 cells/well were enough to detect a significant signal, while 10,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-CDK1 (Tyr15) assay was 8 times more sensitive than the Western Blot technique.

HTRF phospho CDK1 (Tyr15) assay compared to Western Blot

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In collaboration with Bayer - Présentations scientifiaues

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Cisbio Product Catalog 2019

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A guide to Homogeneous Time Resolved Fluorescence

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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. - Notes techniques

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Oncology Guide

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