Phospho-CDK2 (Tyr15) Cellular Kit
Simple and robust detection kit for Phospho-CDK2 (Tyr15)
The Total Cyclin D1 kit is designed to monitor the expression level of cellular Cyclin D1, an oncogene overexpressed in many human cancers.
The Total Cyclin D1 cell-based assay conveniently and accurately detects the levels of total Cyclin D1 within cells. This kit is compatible with the buffers from the phospho-Cyclin D1 (Thr286) kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
Cyclin D family members (including Cyclin D1, Cyclin D2, and Cyclin D3) are important regulators of cell cycle progression. They are integral mediators of growth factor-dependent G1 to S phase progression by playing the role of allosteric regulators of CDK4/6.
Cyclin D1 is upregulated in presence of extracellular mitogenic stimuli, and is then rapidly downregulated by proteasomal degradation following its phosphorylation on Thr286 by the kinase GSK-3β.
Cyclin D1 is an oncogene which is overexpressed in many human cancers. Abnormal increased levels of Cyclin D1 result in dysregulated CDK4/6 activity, and lead to tumorigenesis. Cyclin D1 overexpression may be caused by gene amplification or chromosomal rearrangements, or be a result of impaired degradation of the protein.
The Total Cyclin D1 assay quantifies the expression level of Cyclin D1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total Cyclin D1 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 D1 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 D1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Total Cyclin D1 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.
MCF7 cells were plated in a 96-well plate (50,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs targeting specifically Cyclin D1, Cyclin D2, or Cyclin D3, as well as with a non-targeting siRNA used as negative control. Following a 24h incubation with siRNAs, the medium was renewed for an additional 24h incubation, and the cells were then treated with 10 µM MG132 for 4h. After cell lysis, 16 µL of lysates were transferred into a low volume white microplate and 4 µL of the HTRF Total Cyclin D1 detection antibodies were added to detect Total Cyclin D1. In parallel, 4 µL of the same lysates were transferred into separate wells of the detection plate to detect GAPDH using the HTRF GAPDH Housekeeping assay (Cat # 64GAPDHPEG, 64GAPDHPEH). The HTRF signal was recorded after an overnight incubation for both assays.
Cell transfection with the siRNA targeting Cyclin D1 led to a 78% signal decrease on the Total Cyclin D1 assay compared to the cells transfected with the non-targeting siRNA. On the contrary, the knockdown of Cyclin D2 and Cyclin D3 did not induce any signal decrease. Furthermore, the levels of the housekeeping protein GAPDH were not significantly affected by the treatment with siRNAs, demonstrating that the signal decrease observed for the Total Cyclin D1 assay in presence of Cyclin D1 siRNA was not caused by cell cytotoxicity. Taken together, this data shows that the HTRF Total Cyclin D1 assay is specific for Cyclin D1 and does not cross-react with other Cyclin D family members.
Different cell densities of the human fibrosarcoma cell line HT-1080 were plated in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37 °C-5% CO2. The cells were treated or not with 10 µM MG132 for 4 hours, and then lysed with 50 µL of supplemented lysis buffer #1 (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 HTRF Phospho-Cyclin D1 (Thr186) or Total Cyclin D1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As expected, the levels of Phospho-Cyclin D1 (Thr286) and Total Cyclin D1 increased in presence of the proteasome inhibitor MG132, which blocks the ubiquitin-dependent proteasomal degradation of the protein and induces its accumulation in the cells.
The human breast cancer cell line MCF7 was plated in complete culture medium in a 96-well culture-treated plate under 100,000 cells/well, and incubated overnight at 37 °C-5% CO2. The cells were treated with increasing concentrations of Everolimus for another overnight incubation, and then lysed with 50 µL of supplemented lysis buffer #1 (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 D1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Cell treatment with the mTORC1 inhibitor Everolimus induced a dose-dependent decrease in the cellular content of Cyclin D1, caused by its degradation. These results are in accordance with the literature (Chen et al., Am J Physiol Cell Physiol 317: C244–C252, 2019).
MCF7 cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were treated with 10 µM MG132 for 4h, and then 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 Cyclin D1 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.
Using the HTRF Total Cyclin D1 assay, 400 cells/well were enough to detect a significant signal, while 12,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF Total Cyclin D1 assay was 32 times more sensitive than the Western Blot technique.
Physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
64CYCD1TPEG-64CYCD1TPEH - Product Insert
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