Revvity, 64PLKT2PEG, HTRF Human and Mouse Phospho-PLK1 (Thr210) Detection Kit, 500 Assay Points

Overview
This HTRF cell-based assay enables the rapid, quantitative detection of PLK1 phosphorylated at Threonine 210. Polo-like kinase 1 (PLK1) is the principle member of the well conserved serine/threonine kinase family and is essential for cell division (mitosis). As such, it is a key regulator of the G2/M checkpoint of the cell cycle
PLK1 activation for cell cycle regulation: PLK1 is activated by phosphorylation on Thr210 by AurA kinase. Activated PLK1 phosphorylates CDC25.
PLK1 is a critical protein responsible for mitosis and cell cycle progression/cell proliferation. This makes it an attractive cancer treatment target.
How it works
Phospho-PLK1 (Thr210) assay principle
The HTRF Phospho-PLK1 (Thr210) assay measures PLK1 when phosphorylated at Thr210. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-PLK1 (Thr210) 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, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies and 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-PLK1 (Thr210 ) 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 the Phospho-PLK1 (Thr210) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-PLK1 (Thr210 ) one-plate assay protocol
Detection of Total PLK1 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 4
Molecular Modification-Phosphorylation
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