Skip to main content

HTRF Human and Mouse Total CDK2 Detection Kit, 500 Assay Points

The Total CDK2 kit is designed to monitor the expression level of cellular CDK2 (Cyclin-Dependent Kinase 2), and can be used as a normalization assay for the Phospho-CDK2 (Tyr15) kit.

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 & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Part number: 64CDK2TPEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
Your price:
USD 2,147.00
USD 2,147.00 /each
Part number: 64CDK2TPEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price:
USD 0.00
USD 12,490.00 /each

Overview

The Total CDK2 cellular assay monitors total CDK2, and can be used as a normalization assay with our phospho-CDK2 (Tyr15) kit. This kit is compatible with the buffers from the phospho-CDK2 kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.

CDK2 (Cyclin-Dependent Kinase 2) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (including also CDK1, CDK4, and CDK6). CDK2 is activated by interaction with Cyclin E and Cyclin A during the late G1 phase and the S phase respectively, and is also regulated by two major phospho-sites. Phosphorylation at Tyr15 by the kinase Wee1 is inhibitory by preventing ATP binding, whereas Tyr15 dephosphorylation by the phosphatase Cdc25A and phosphorylation at Thr160 by CAK (CDK Activating Kinase) is required for its full activity.

CDK2 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 CDK2.

Specifications

Assay Points
500
Assay Target Type
Kit
Assay Technology
HTRF
Brand
HTRF
Quantity
1
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

How it works

Total CDK2 assay principle

The Total CDK2 assay quantifies the expression level of CDK2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK2 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of CDK2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.

Principle of the HTRF total CDK2 assay

 

Total CDK2 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 Total CDK2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF total CDK2 assay

 

Total CDK2 one-plate assay protocol

Detection of Total CDK2 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 total CDK2 assay

 

Assay validation

Validation of the specificity of Total CDK2, Total CDK4, and Total CDK6 assays by siRNA experiments

HeLa and HEK293 cells were plated in 96-well plates (40,000 and 50,000 cells/well respectively) and cultured for 24h. The cells were then transfected with siRNAs specific for CDK1, CDK2, CDK4, or CDK6, as well as with a negative control siRNA. After 48h incubation, the cells were lyzed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total CDK2, Total CDK4, or Total CDK6 detection antibodies. The HTRF signal was recorded after an overnight incubation.

Cell transfection with each specific CDK2, CDK4, or CDK6 siRNA led to 77 to 97% signal decrease compared to the cells transfected with the negative siRNA. It should be noted that the small signal decrease observed on total CDK6 assay when cells were transfected with CDK2 siRNA was expected, since CDK2 knockdown leads to down-regulation of CDK6 (Bačević et al., Sci Rep. 2017; 7: 13429). Taken together, these data demonstrate that HTRF Total CDK2, Total CDK4, and Total CDK6 assays are specific for each kinase and do not cross-react with other cell cycle CDK family members.

total-CDK2-cellular-kit

 

total-CDK2-cellular-kit

 

total-CDK2-cellular-kit

 

Dephosphorylation of CDK2 Tyr15 by alkaline phosphatase

HeLa cells were cultured in a T175 flask for 48h and treated with 0.5 µg/mL Aphidicolin for 20h. The cells were then lyzed with 3 mL of supplemented lysis buffer #2 (1X) and only a part of the lysate was treated with alkaline phosphatase (AP). For the detection step, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-CDK2 (Tyr15) or Total CDK2 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, alkaline phosphatase induced the nearly complete dephosphorylation of CDK2 on Tyr15, while the total level of the kinase was not modulated by the treatment.

Dephosphorylation of CDK2 Tyr15 by alkaline phosphatase in HeLa cells

 

Phospho-CDK2 (Tyr15) modulation using cell cycle blockers

Human HeLa cells and mouse NIH-3T3 cells were cultured in a 96-well plate (50,000 cells/well) for 24h and then treated for 16h with the cell cycle blockers Aphidicolin and Nocodazole respectively.

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-CDK2 (Tyr15) or Total CDK2 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, Aphidicolin (which arrests cells in the early S phase) induced a dose-dependent accumulation of phosphorylated CDK2 at the inhibitory site Tyr15.

Inversely, Nocodazole (which arrests cells at the G2/M border) induced a dose-dependent decrease in CDK2 phosphorylation on Tyr15.

The expression level of the kinase remained relatively stable, whatever the cell treatment.

Modulation of phospho-CDK2 (Tyr15) by Aphidicolin in HeLa cells

 

Modulation of phospho-CDK2 (Tyr15) by Nocodazole in NIH-3T3 cells

 

Phospho-CDK2 (Tyr15) inhibition using Wee1 kinase inhibitors

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-CDK2 (Tyr15) or Total CDK2 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 CDK2 at Tyr15, while the expression level of the protein was not modulated by the treatments.

Inhibition of phospho-CDK2 (Tyr15) by Adavosertib in HeLa cell
Inhibition of phospho-CDK2 (Tyr15) by PD0166285 in HeLa cells
HTRF total CDK2 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After 48h incubation, the cells were lyzed with 3 mL of supplemented lysis buffer #2 (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 total CDK2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total CDK2 assay, 800 cells/well were enough to detect a significant signal, while 1,600 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF total CDK2 assay was twice as sensitive as the Western Blot technique.

Comparison of HTRF total CDK2 kit with western blot

 

Simplified pathway

Role of CDK2 in the cell-division cycle

CDK2 (Cyclin-Dependent Kinase 2) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (also including CDK1, CDK4, and CDK6).

Mitogenic signals, such as growth factors, trigger cells to enter the G1 phase of the cell cycle by inducing cyclin D synthesis, leading to the formation of active CDK4/6-cyclin D complexes. CDK4 and CDK6 mono-phosphorylate the protein of retinoblastoma (RB), which still binds to transcription factor E2F, but some genes can be transcribed, such as cyclin E. In the late G1 and early S phases, Cyclin E interacts with and activates CDK2, which in turn phosphorylates additional sites on RB resulting in its complete inactivation. The E2F-responsive genes required for S phase progression are thus induced, such as Cyclin A which then interacts with CDK2 to form Cyclin A/CDK2 complexes. Activated CDK2 finally phosphorylates Cdc25B & Cdc25C phosphatases, which in turn activate CDK1, required for progression in the G2 and M phases of the cell-division cycle.

Total CDK2 signaling pathway

 

Resources

1-2 of 2 Resources
Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

Guide
Protein degradation awareness in a single guide

<p>An in-depth review of molecular and cellular pathways</p><p>The maintenance of proteostasis, the biological mechanisms that control the...</p>

SDS, COAs, Manuals and more Illuminator

SDS, COAs, Manuals and more

Are you looking for technical documents for this product. We have housed them in a dedicated section., click on the links below to explore.

Scroll Icon