Revvity, 64CDK5TPEG, HTRF Human and Mouse Total CDK5 Detection Kit, 500 Assay Points

Overview
Cdk5 belongs to Cyclin-dependent kinases (Cdks) family. Despite having a high sequence homology with other members of Cdks family, CDK5 is an unusual member because its not directly involved in the regulation of the cell cycle and its activation does not require the phosphorylation state or binding with a cyclin.
CDK5 represents an attractive pharmacological target as its dysregulation is implicated in various neurodegenerative diseases such as Alzheimer's, Parkinson's and various types of cancers.
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 CDK5 assay principle
The Total CDK5 assay quantifies the expression level of CDK5 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 CDK5 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 CDK5 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 CDK5 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total CDK5 one-plate assay protocol
Detection of Total CDK5 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-500 assay points