Total Cyclin E1 assay principle

The Total Cyclin E1 assay quantifies the expression level of Cyclin E1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total Cyclin E1 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 Cyclin E1 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 Cyclin E1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate before the addition of Total Cyclin E1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total Cyclin E1 one-plate assay protocol

Detection of Total Cyclin E1 with HTRF reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Specificity of HTRF (h) Total Cylin E1 assay using siRNA experiments

HeLa cells were plated in 96-well plates (50,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for Cyclin E1 and Cyclin E2, 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 Cyclin E1 detection antibodies. The HTRF signal was recorded after 3 hours of incubation.

Cell transfection with Cyclin E1 siRNA led to an 80% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed for cells transfected with Cyclin E2 siRNA. The data demonstrate that the HTRF Total Cyclin E1 assay is specific for the detection of Cyclin E1 protein and does not cross-react with Cyclin E2.

HTRF (h) Total Cyclin E1 assay versatility on human cell lines

The adherent Human cell lines HeLa, MCF7, U20S, or HAP1 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 Cyclin E1 detection reagents. The HTRF signal was recorded after 3 hours of incubation.

The HTRF Total Cyclin E1 assay efficiently detects Cyclin E1 in various cellular models expressing different levels of the protein.

HTRF Total Cyclin E1 assay compared to Western Blot

U2OS 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 #2 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer#2, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total Cyclin E1 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 Cyclin E1 assay was 16-fold more sensitive than the Western Blot technique.

Cyclins play a key role in the orderly progression of the cell division cycle through their timed expression and their ability to bind, activate and enhance the substrate affinity of their associated cyclin-dependent protein kinases (CDKs). Cyclin E1 and Cyclin E2 are activators of cyclin-dependent kinase 2 (CDK2). In mammalian cells, the mitogen-stimulated activities of CDK4 or CDK6 in association with a D-type cyclin promote transcription of cyclin E during the G1 phase and activation of CDK2/cyclin E kinase activity. Levels of Cyclin E decline during the S phase, in response to auto- and other phosphorylation events that target Cyclin E for ubiquitination and degradation by the proteasome. Cyclin E1 plays a direct role in the initiation of DNA replication , the control of histone biosynthesis, and the centrosome cycle. Cyclin E1 is associated with disease progression in various malignancies, and has been clinically associated with a poor prognosis.