Revvity, 64CDK8TPEH, HTRF Human and Mouse Total CDK8 Detection Kit, 10,000 Assay Points

Overview
CDK8 and its paralog, CDK19, also named "Mediator Kinase" are cyclin-dependent kinases that have been implicated in cellular homeostasis and developmental programming. CDK8 and CDK19 are incorporated, in a mutually exclusive manner, as part of a 4-protein complex called the Mediator kinase module. This module reversibly associates with the Mediator, a 26 subunit protein complex that regulates RNA Polymerase II mediated gene expression. CDK8 has been implicated in the development of different human diseases, and in particular cancer.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
Total CDK8 assay principle
The Total CDK8 assay quantifies the expression level of CDK8 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The 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 CDK8 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 CDK8 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 HTRF Total CDK8 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total CDK8 one-plate assay protocol
Detection of Total CDK8 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 2 Lysis Buffer 3 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
Unit Size-10,000 assay points