Revvity, 64CDK2TPEH, HTRF Human and Mouse Total CDK2 Detection Kit, 10,000 Assay Points

Overview
The Total CDK2 cellular assay monitors total CDK2, and can be used as a normalization assay with our phospho-CDK2 (Tyr15) kit. This kit is compatible with the buffers from the phospho-CDK2 kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
CDK2 (Cyclin-Dependent Kinase 2) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (including also CDK1, CDK4, and CDK6). CDK2 is activated by interaction with Cyclin E and Cyclin A during the late G1 phase and the S phase respectively, and is also regulated by two major phospho-sites. Phosphorylation at Tyr15 by the kinase Wee1 is inhibitory by preventing ATP binding, whereas Tyr15 dephosphorylation by the phosphatase Cdc25A and phosphorylation at Thr160 by CAK (CDK Activating Kinase) is required for its full activity.
CDK2 inhibitory phosphorylation at Tyr15 is essential for maintaining genome integrity and preventing DNA damage during the S phase. The Wee1/Cdc25A axis is therefore an attractive target for cancer therapy and may represent a unique approach to sensitize cancer cells with hyperactive CDK2.
How it works
Total CDK2 assay principle
The Total CDK2 assay quantifies the expression level of CDK2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK2 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 CDK2 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 CDK2 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total CDK2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total CDK2 one-plate assay protocol
Detection of Total CDK2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 2 Lysis Buffer 3
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-10,000 assay points