This Raf-1 Kinase Assay Kit is Intended for the assay of recombinant Raf-1 (supplied) kinase activity, with experimental variables as determined by the end user. The assay kit is designed to measure Raf-1 dependent phosphotransferase activity in a kinase reaction using recombinant MEK1, unactive as a Raf-1 substrate:
Active Raf-1, 30 degrees C, 30mins.
MEK1 (unactive) + ATP --------------------------------------------> Active MEK1 + ADP
The enzyme assay is rapid, convenient and specific for the Raf family of kinases. Each kit contains sufficient reagents for 50 individual Raf-1 assays.
R0495-14A1 Raf-1 (truncated), active: 1 x10ug. Concentration: Lot-specific. Supplied as a liquid in 50mM Tris-HCl, pH 7.5, 270mM sucrose, 150mM NaCl, 0.1mM EGTA, 0.1% 2-mercaptoethanol, 0.03 % Brij-35, 1mM benzamidine and 0.2mM PMSF. N-terminal GST-tagged recombinant human c-Raf residues 306 to end, containing the mutations Y340D and Y341D. Purity: 84% by SDS-PAGE and Coomassie blue staining. MW: 65kD. Specific activity: Lot-specific. 1 Unit of Raf-1 is equal to 1 Unit of MAPK2 activity which in turn is equal to 1nmole of phosphate incorporated into 0.33mg/ml myelin basic protein (MBP) per minute at 30 degrees C with a final ATP concentration of 100uM.
R0495-14A2 MEK1, unactive:1x50ug. Concentration: Lot-specific. Supplied as a liquid in 50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1mM EGTA, 0.03% Brij-35, 5% glycerol, 0.1% 2-mercaptoethanol, 0.2mM PMSF, 1mM benzamidine. N-terminal GST and C-terminal 6His-tagged, recombinant full-length human MEK1 expressed in E. coli. MW: 71kD. Purity: 97% by SDS-PAGE and Coomassie blue staining. Maximally activated with B-Raf, Raf-1, or MEKK. Activated by phosphorylation with Raf-1 (truncated).
R0495-14A3 Assay Dilution Buffer I (ADBI): 1x1ml. Supplied as a liquid in 20mM MOPS, pH 7.2, 25mM-glycerol phosphate, 5mM EGTA, 1mM sodium orthovanadate, 1mM dithiothreitol.
R0495-14A4 Magnesium/ATP Cocktail: 1x1ml. Supplied as a liquid in 75mM magnesium chloride and 500uM ATP in ADBI.
R0495-14A5: Anti-phospho MEK1 (Ser218/222)/MEK2 (Ser222/226). 1x200ug. Concentration: 1mg/ml. Protein A purified rabbit IgG supplied as a liquid in 0.07M Tris-glycine, pH 7.4, 0.105M NaCl, 0.035% sodium azide, and 30% glycerol. Specificity: Recognizes MEK1/MEK2, Mr 45kD. A non-specific protein may also be detected in HEK293 cells, Mr 50kD. Species Crossreactivity: human, mouse and rat.
Storage and Stability:
Store R0495-14A3, R0495-14A4 and R0495-14A5 at -20 degrees C. Store R0495-14A1 and R0495-14A2 at -70 degrees C. Kit components are Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.