HTRF Human Total CDK1 Detection Kit HTRF®

The Total CDK1 assay quantifies the expression level of CDK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK1 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 CDK1 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.

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 CDK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

HeLa cells were plated in 96-well plates (10,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, or CDK12, 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 before the addition of 4 µL of the HTRF Total CDK1 detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #11) was also transferred into the microplate to monitor the GAPDH level using the GAPDH Housekeeping Cellular Kit (64GAPDHPET/G/H). HTRF signals for both kits were recorded after an overnight incubation.

Cell transfection with CDK1 siRNA led to a 81% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed with cells transfected with CDK2, CDK4, CDK6, CDK7, CDK9, or CDK12 siRNAs. The  level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrate that the HTRF Total CDK1 is specific for the detection of the CDK1 protein,and does not cross-react with other CDK family members.

Various human cell lines, either adherents HeLa,MCF7, SHSY5Y cells or suspension such as K562 cells, were seeded at 100,000 cells / well in a 96-well microplate. After 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 Total CDK1 detection reagents. The HTRF signal was recorded after an overnight incubation.

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were lysed 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 total CDK1 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total CDK1 assay, 1250 cells/well were enough to detect a significant signal,  while 10,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF total CDK1 assay is 8 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).