Revvity, 64BRAFTPEH, HTRF Human Total B-RAF Detection Kit, 10,000 assay points

Overview
B-RAF is a proto-oncogene protein kinase. The RAS-RAF-MEK signaling cascade plays a pivotal role in modulating cellular processes such as proliferation, differentiation, and survival. This pathway is often constitutively activated in human malignancies characterized by RAS or RAF oncogenic drivers. Developing chemotherapeutic cRAF inhibitors is an attractive area of research in oncology and infectious diseases.
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 B-RAF assay principle

The Total B-RAF assay measures B-RAF levels in cells. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two antibodies, one with a donor fluorophore and the other with an acceptor. In the presence of B-RAF, labeled antibodies form an immune complex, bringing the donor fluorophore close to the acceptor and generating a FRET signal. The signal intensity is directly proportional to the concentration of total protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.
 

 
Total B-RAF 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 B-RAF HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored

 
Total B-RAF one-plate assay protocol

Detection of Total B-RAF with HTRF reagents can be performed on a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-B-RAF
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points