Raf1, Truncated, active, Recombinant, Human

N-terminal GST-tagged recombinant human Raf-1 residues 306-end, containing the mutations Y340D and Y341D. Expressed by baculovirus in Sf21 insect cells. S226 of the recombinant protein is equivalent to S306 of native sequence. Y340 and Y341 of the native Raf-1 sequence have been mutated to aspartate to provide constitutive activation of the enzyme. Recombinant Raf1 sequence: 1 MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID , 61 GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL DIRYGVSRIA YSKDFETLKV , 121 DFLSKLPEML KMFKDRLCHK TYLNGDHVTH PDFMLYDALD VVLYMDPMCL DAFPKLVCFK , 181 KRIEAIPQID KYLKSSKYIA WPLQGWQATF GGGDHPPKSD LVPRGSQPKT PVPAQRERAP , 241 VSGTQEKNKI RPRGQRDSSD DWEIEASEVM LSTRIGSGSF GTVYKGKWHG DVAVKILKVV , 301 DPTPEQFQAF RNEVAVLRKT RHVNILLFMG YMTKDNLAIV TQWCEGSSLY KHLHVQETKF , 361 QMFQLIDIAR QTAQGMDYLH AKNIIHRDMK SNNIFLHEGL TVKIGDFGLA TVKSRWSGSQ , 421 QVEQPTGSVL WMAPEVIRMQ DNNPFSFQSD VYSYGIVLYE LMTGELPYSH INNRDQIIFM , 481 VGRGYASPDL SKLYKNCPKA MKRLVADCVK KVKEERPLFP QILSSIELLQ HSLPKINRSA, 541 SEPSLHRAAH TEDINACTLT TSPRLPVF, Specific Activity: ~50,000U/mg (lot-specific) , Unit Definition:, 1u Raf1 activity = 1u MAPK2 activity which in turn is equal to 1nmole of phosphate incorporated into 0.33mg/ml myelin basic protein (MBP) per minute at 30°C with a final ATP concentration of 100uM. Note the activity is determined using a triple linked assay which involves the activation of MEK1 by RAF-1, followed by the subsequent activation of MAPK2 by the activated MEK1. Storage and Stability: Store at -70°C. Stable for 6 months. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Handling Recommendations: Rapidly thaw the vial under cold water and immediately place on ice. Aliquot unused material into pre-chilled microcentrifuge tubes and immediately snap-freeze the vials in liquid nitrogen prior to re-storage at - 70°C.