Description
The Phospho-c-RAF or Raf-1 (Ser338) Cellular Assay Kit is designed for the robust quantification of phoshorylated c-RAF at Ser-338 directly using a streamlined mix-and-read, no-wash protocol. c-RAF 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
Phospho-c-RAF (Ser338) Assay principle
The Phospho-c-RAF (Ser338) assay measures c-RAF when phosphorylated at Ser338. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-c-RAF (Ser338) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent 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 proteins phosphorylation state under a no-wash assay format.
Phospho-c-RAF (Ser338) 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 phospho-c-RAF (Ser338) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-c-RAF (Ser338) 1-plate assay protocol
Detection of Phosphorylated c-RAF (Ser338) 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.
