The phospho-cRaf (Ser338) kit enables the cell-based detection of cRaf phosphorylation on Serine 338 as a readout of MAPK pathway.
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  • Ease-of-use
  • Highly specific
  • High sensitivity
The phospho-cRaf (Ser338) kit enables the cell-based detection of cRaf phosphorylation on Serine 338 as a readout of MAPK pathway.
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Overview

The Phospho-c-RAF (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.

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 protein’s 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.
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