Revvity, 64CYCD1T6PEG, HTRF Human Phospho-Cyclin D1 (Thr286) Detection Kit, 500 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately detects phosphorylated Cyclin D1 at Thr286. ​​​​​​​
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 whose overexpression results in dysregulated CDK4/6 activity and leads to tumorigenesis. Cyclin D1 overexpression may be caused by gene amplification or chromosomal rearrangements, or as a result of impaired degradation of the protein. Mutations and deletions affecting its phosphorylation on Thr286 have been reported in several human cancers.
How it works
Phospho-Cyclin D1 (Thr286) assay principle
The Phospho-Cyclin D1 (Thr286) assay measures Cyclin D1 when phosphorylated at Thr286. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-Cyclin D1 (Thr286) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

 

Phospho-Cyclin D1 (Thr286) 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 adding Phospho-Cyclin D1 (Thr286) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-Cyclin D1 (Thr286) one-plate assay protocol
Detection of phosphorylated Cyclin D1 (Thr 286) 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 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-500 assay points