Revvity, 64EGFRL8PEH, HTRF Human Total EGFR L858R Detection Kit, 10,000 Assay Points

Overview
Drug resistance in patients with EGFR mutated non-small cell lung cancer treated with tyrosine kinase inhibitors (TKIs) is one of the main challenges for cancer targeted therapy. Designing specific PROTAC compounds to degrade EGFR mutated proteins is expected to offer a promising approach to treating such patients, and overcome drug resistance.
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-EGFR L858R assay principle
The HTRF Total-EGFR L858R assay quantifies the expression level of mutated EGFR L858R in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-EGFR Mutant L858R 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 EGFR Mutant L858R 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-EGFR L858R 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 Total-EGFR L858R HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored
 

 

Total-EGFR L858R one-plate assay protocol
Detection of Total-EGFR Mutant L858R 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
Technology-TR-FRET
Unit Size-10,000 assay points