• Affinity reagent for the detection of activated (GTP-bound) Ras protein in cell and tissue lysates

The Ras GTP Binding Domain (RBD) of human Raf protein kinase has been produced in a bacterial expression system. The recombinant protein consists of amino acids 51-149 of Raf kinase and is tagged with GST (28 kDa) at its amino terminus. The approximate molecular weight of the Raf-RBD GST protein is 35 kDa. RF02 beads (2000 µg protein per tube) are supplied as a pink/purple lyophilized powder, each tube is sufficient for approximately 20 assays.

Protein purity is assessed by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gel. Purity was determined to be ≥90% pure.

Raf-RBD beads specifically recognize and bind the active (GTP-bound) form of Ras (N-, H-, and K-Ras), with minimal affinity for the inactive (GDP-bound) form. A standard assay (30 µl beads, 200 µl lysate) demonstrates this selectivity by comparing GTPγS-loaded bovine brain lysate to GDP-loaded lysate. After isolating activated Ras, western blotting with a pan-Ras antibody is performed. GTPγS, a non-hydrolysable GTP analog, locks Ras in its active conformation. Running GDP and GTPγS conditions in parallel validates both the specificity and performance of the beads. This assay results in a 3-5X enhancement of active Ras signal in the GTP γS sample.