HTRF Human Total CDK7 Detection Kit, 10,000 Assay Points

Total CDK7 assay principle

The Total CDK7 assay quantifies the expression level of CDK7 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK7 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 CDK7 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.

Total CDK7 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 CDK7 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total CDK7 one-plate assay protocol

Detection of Total CDK7 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.

Assessment of total CDK7 levels in human cell lines

Various human cell lines, either adherent HeLa, MCF7, or SHSY5Y cells, or suspension, such as K562 cells, were seeded at 100,000 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 Total CDK7 detection reagents. The HTRF signal was recorded after an overnight incubation.

HTRF total CDK7 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 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 CDK7 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF Total CDK7 assay, 1250 cells/well were enough to detect a significant signal, while 5,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF Total CDK7 assay is 4 times more sensitive than the Western Blot technique.

Role of CDK7 in the cell-division cycle

CDKs are traditionally separated into 2 subfamily members: CDKs that coordinate cell cycle progression (CDK1, CDK2, CDK4, and CDK6) and transcriptional CDKS (CDK7, CDK8, CDK9, CDK12, and CDK13).

CDK7 (cyclin dependant kinase 7 ) is both a CDK-activating kinase (CAK), which phosphorylates cell-cycle CDKs (CDK1-CDK2-CDK4 and CDK6), and a component of the general transcription factor TFIIH, which regulates the initiation of the transcription through phosphorylation of the C-terminal domain (CTD) of the RNA Pol II.