Revvity, 64CRAFS43V2PEH, HTRF Phospho-c-RAF (Ser43) Detection Kit, 10,000 Assay Points

Overview
The Phospho-c-RAF or Raf-1 (Ser43) Cellular Assay kit is designed for the robust quantification of phoshorylated cRaf at Ser-43 directly using a streamlined mix-and-read, no-wash protocol. cRaf plays a pivotal role in cell proliferation and differentiation. This kit can be used from basic research to high throughput drug screening. Its versatility means it is suitable for many applications, ranging from basic research to the analysis of pharmacological questions in cellular models, and has applications in oncology and infectious disease research.
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-cRAF (Ser43) assay principle
The Phospho-cRAF (Ser43) assay measures cRAF when phosphorylated at Ser43. 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, and 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-cRAF (Ser43) 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 Phospho-cRAF (Ser43) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-cRAF (Ser43) one-plate assay protocol
Detection of Phosphorylated cRAF (Ser43) 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 facilitates miniaturization while maintaining HTRF quality. Application-Cell Signaling
Brand-HTRF
Buffer/Solvent-Lysis Buffer 2
Detection Modality-HTRF
Host Species-Human
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-cRAF
Target Class-Phosphoproteins
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points