Total CDK5 assay principle

The Total CDK5 assay quantifies the expression level of CDK5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The 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 CDK5 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 CDK5 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 HTRF Total CDK5 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total CDK5 one-plate assay protocol

Detection of Total CDK5 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.

Specificity of Total CDK5 using HAP1-KO cells

The CDK5 expression level was assessed with the HTRF Total CDK5 kit in Wild type HAP1 (WT) and CDK5 knockout HAP1 cell lines. The cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The different cell lines were cultured in a 96-well plate for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X), then 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

In HAP1 KO CDK5 cells, the HTRF signal was equivalent to the non-specific signal, indicating complete CDK5 gene silencing, whereas the CDK5 level was well detected in the wildtype cell line, as expected. This result demonstrates the selectivity of the HTRF CDK5 kit in detecting only CDK5 protein.

Total CDK5 assay detection in various cell lines

The adherent Human cell lines HeLa, U87-MG, HCT116, and SHSY5Y or Neuro2A mouse 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 CDK5 detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

The HTRF Total CDK5 assay efficiently detects CDK5 in various cellular models expressing different levels of the protein.

HTRF (h/m) Total CDK5 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 and cell medium removal, 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#1, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total CDK5 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total CDK5 assay was 32-fold more sensitive than the Western Blot technique.

CDK5 Signaling Pathways

Cyclin-dependent kinases (CDKs) are a group of serine/threonine protein kinases which regulate post-mitotic processes such as neuronal activity, neuronal migration during development, and neurite outgrowth. Activation of CDK5 requires its association with a regulatory subunit p35. p35 is a short-lived protein, and its phosphorylation by CDK5 targets leads to ubiquitin-mediated proteolysis. Calpain1 also directly cleaves p35 to release a 25 kD protein product, which causes prolonged activation and mislocalization of CDK5 and hyperphosphorylation of Tau.

In addition, CDK5 activation promotes DNA damage by DSBs and consequently leads to ncreased levels of cell cycle regulators such as cyclins and pRb by the activation of DNA damage responses. CDK5 also responds to DNA damage by phosphorylating ATM, which leads to cell death through p53. CDK5 phosphorylates other substrates such as Rb, Akt, p21, STAT3, and CDK1, all of which are involved in cell cycle progression, contributing to aberrant cell cycle activation.