Raf-RBD domain protein agarose beads (RF02)

Material
The Ras binding domain (RBD) of the c-Raf kinase protein (Raf) protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, Ras proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Ras as well as to specifically block the activity of Ras in vitro and in vivo.

The protein is supplied in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. This product is used in our Ras activation assay Biochem Kit™ (Cat. # BK0 08). The GST-tagged Raf-RBD protein can be released from the beads by incubation with 10 mM reduced glutathione.

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Raf-RBD protein is >80% pure (see Figure 2).

Figure 2: GST-Raf-RBD protein purity determination. A 20 µg sample of RF02 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-Raf-RBD protein runs at approximately 35 kDa.

Biological Activity
Raf-RBD protein specifically recognizes and binds the active, GTP-bound, forms of Ras proteins. It has a much lower affinity for the inactive, GDP-bound, form Ras. When coupled to a colored glutathione sepharose matrix, the Raf-RBD protein beads become a convienent tool for assaying the activity of the Ras proteins. The quality control biological assay for Raf-RBD protein beads consists of a Ras protein pulldown frombovine brain extracts loaded with either GTPγS (Cat. # BS01) or GDP.

Examples of publications where this product was used:
Zhang, J., Anastasiadis, P. Z., Liu, Y., Thompson, E. A. and Fields, A. P. (2004). Protein kinase C (PKC) βII induces cell invasion through a Ras/Mek-, PKC ι/Rac 1-dependent signaling pathway. J. Biol. Chem. 279, 22118-22123.