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.

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.

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.

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.

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.

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.

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.

CDK1 (Cyclin-Dependent Kinase 1) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (also including CDK2, 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 (centrosome maturation and separation, chromosome condensation and mitotic entry after nuclear envelope breakdown).