Revvity, 64CDK4TPEH, HTRF Human and Mouse Total CDK4 Detection Kit, 10,000 Assay Points

Overview
The Total CDK4 cellular assay monitors CDK4 protein levels.
CDK4 (Cyclin-Dependent Kinase 4) is a member of the subfamily of CDKs that coordinate cell cycle progression in mammalian cells (including also CDK1, CDK2, and CDK6). In the early G1 phase of the cell cycle, CDK4 is activated by interaction with Cyclin D, and mono-phosphorylates the tumor suppressor RB (protein of retinoblastoma), leading to the transcription of genes required for G1/S phase transition.
Dysregulated activation of CDK4, leading to uncontrolled cell division, is a hallmark of cancers. Inhibition of CDK4 is thus an active research area, especially since the emergence of new approaches involving PROTACs (PROteolysis TArgeting Chimeras).
How it works
Total CDK4 assay principle
The Total CDK4 assay quantifies the expression level of CDK4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total CDK4 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 CDK4 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 CDK4 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 CDK4 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total CDK4 one-plate assay protocol
Detection of Total CDK4 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 1 Lysis Buffer 4 Lysis Buffer 5
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