Revvity, 64NSRPEG, HTRF Human and Mouse Total c-Src Detection Kit, 500 Assay Points

Overview
The Total c-Src cellular assay monitors total c-Src and is used as a normalization assay with the phospho-c-Src kit. This protein is a member of the Src kinase family and shares a high degree of sequence identity with the ten other members. This protein is a membrane-associated, non-receptor tyrosine kinase. Its autophosphorylation on the residue Tyr419 indicates its full activation. c-Src kinase is over-expressed and highly activated in many human cancers, making it a promising target for cancer therapy.
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-c-Src assay principle
The Total-c-Src assay quantifies the expression level of c-Src in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-c-Src assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of c-Src 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 proteins expression under a no-wash assay format.

 

Total-c-Src 2-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 adding Total-c-Src HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-c-Src 1-plate assay protocol
Detection of total cSrc 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 3 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-500 assay points