Revvity, 64COFS3PEH, HTRF Human and Mouse Phospho-Cofilin (Ser3) Detection Kit, 10,000 Assay Points

Overview
The kit is designed for the rapid detection of Phospho Cofilin Ser3 in cell supernatant and whole cells. Actin-binding proteins are abundant cellular proteins that regulate cell function by mediating actin polymerization and remodeling. Cofilin, known as a regulator of actin filament dynamics, is a small ~21kDA protein that is ubiquitously expressed in all vertebrates and freely diffuses in eukaryotic cells. Phosphorylation at Ser-3 by kinases attenuates cofilinâ's actin- binding activity. In contrast, dephosphorylation at Ser-3 enhances cofilin-induced actin depolymerization.
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
Phospho-Cofilin (Ser3) assay principle
The Phospho-Cofilin (Ser3) assay measures Cofilin when phosphorylated at Ser3. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both 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-Cofilin (Ser3) two-plate assay protocol
The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-Cofilin (Ser3) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-Cofilin (Ser3) one-plate assay protocol
Detection of Phosphorylated Cofilin (Ser3) 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 4 Lysis Buffer 5
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-10,000 assay points