Phospho-c-RAF (Ser338) Cellular Assay Kit

Homogeneous cell-based c-RAF assay for an upstream Ras-Raf /MAPK signaling pathway readout

Cisbio's cell-based homogeneous HTRF® phospho-c-RAF assay (Ser338) enables the Ras readout of the Ras-Raf-MEK-ERK core cascade of the MAPK, by allowing quantitative detection of the modulation of the RAF serine/threonine-protein kinase, phosphorylated on serine 338. This MAP3K, also known as Raf-1, is phosphorylated by Ras and, in turn, phosphorylates MEK1/2 in the ERK1/2 pathway. With its strategic role in the Ras-Raf-MEK-ERK core cascade of the MAPK signaling pathway, c-RAF plays a role in cell proliferation and differentiation. This makes c-RAF a significant signaling node involved in  most therapeutic areas, but mainly in oncology, for example melanoma, colorectal, pancreatic or prostate cancers. Raf-1 is also involved in several infectious and rare diseases. The kit is suitable for many applications, ranging from basic research to the analysis of pharmacological questions in cellular models. The simple add-and-read phospho-c-RAF assay protocol eliminates all wash steps, for faster analyses and easy miniaturization, amenable to automation. The phospho-c-RAF (Ser338) ready-to-use kit contains all the reagents you need, and offers enhanced convenience over other immunoassay technologies.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio's phospho-c-RAF assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-c-RAF antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-c-RAF antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The phospho-c-RAF assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, phospho-c-RAF can be quantitatively detected using the HTRF phospho-c-RAF kit reagents and most TR-FRET multimode plate readers.

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For the detection of phosphorylated c-RAF, lysates are subsequently transferred to a 384 small volume assay plate where the HTRF reagents are added. This also enables the monitoring of cell viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
cRaf phospho-S338 kit - 500 tests63ADK018PEG
cRaf phospho-S338 kit - 10,000 tests63ADK018PEH
cRaf phospho-S338 kit - 50,000 tests63ADK018PEY

Companion products

DescriptionCat. noProduct insertMSDS
cRaf phospho-S338 kit - control lysate63ADK018TDA