Revvity, 64CYCD1TPEG, HTRF Human and Mouse Total Cyclin D1 Detection Kit, 500 Assay Points

Overview
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.
How it works
Total Cyclin D1 assay principle
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.

 

Total Cyclin D1 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 D1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total Cyclin D1 one-plate assay protocol
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. Application-Cell Signaling
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-500 assay points