The Total Cyclin E1 kit is designed to monitor the expression level of cellular Cyclin E1, an oncogene overexpressed in many human cancers.
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E-type cyclins play an important role in the transition of quiescent cells into the cell cycle. Two E-type cyclins have been previously described, Cyclin E1 and Cyclin E2. Cyclin E1 is known to activate CDK2 inducing S-phase transition and DNA synthesis associated with mitosis. Overexpression of Cyclin E1 is directly implicated in many cancers.
This kit is compatible with the buffer from Total & phospho-CDK2 kits (‰ 64CDK2TPEG & ‰ 64CDK2Y15PEG), so the same lysate can be used for analyses of the level of Total Cyclin E1, Total CDK2 and CDK2 phospho Tyr15.
Shipping Conditions |
Shipped in Dry Ice
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Unit Size |
500 Assay Points
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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.
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.
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.
HeLa cells were cultured in a 96-well plate (100,000 cells/well) for 6h, and then treated overnight with increasing concentration of Hydroxyurea (inducer of single strand breaks) or Aphidicolin (cell cycle blocker). After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Total Cyclin E1 detection antibodies were added. The HTRF signal was recorded after a 3h incubation.
As expected, the levels of Total Cyclin E1 increased in presence of hydroxyurea or aphidicolin.
HeLa cells were plated in complete culture medium in a 96-well culture-treated plate at 100,000 cells/well, and incubated for 6 hours at 37 °C, 5% CO2. The cells were next treated with increasing concentrations of Nocodazole for 16 hours, and then lysed with 50 µL of supplemented lysis buffer #2 (1X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a low volume white microplate and 4 µL of the Total Cyclin E1 detection reagents were added. The HTRF signal was recorded after 3 hours of incubation.
Cell treatment with Nocodazole (cell cycle blocker) induced a dose-dependent decrease in the cellular content of Cyclin E1.
The Nocodazole treatment had no effect on cell proliferation or viability, as evidenced by the constant ATP levels measured with ATPliteTM assay.
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.
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.
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.
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