Complex of recombinant human mTOR (mechanistic target of rapamycin kinase), recombinant human MLST8 (mTOR associated protein, LST8 homolog), and recombinant human Raptor (regulatory associated protein of mTOR complex 1). The mTOR construct contains an N-terminal Flag-Tag encompassing amino acids 1362-2549(end). The MLST8 construct contains an N-terminal His-tag (6xHis), full length, encompassing amino acids 2-326(end). The Raptor construct contains an N-terminal His-tag (6xHis), full length encompassing amino acids 2-1335(end). Complex was co-expressed and affinity purified.
1. Toral-Barza et al., (2005) Biochem. Biophys. Res. Commun. 24, 332(1):304-10. 2. Rubio-Viqueira and Hidalgo, M. (2006) Adv. Exp. Med. Biol. 587:309-327.
Storage/Stability
At least 6 months at –80°C.
Assay Conditions
Activity was measured using ADP-Glo™ (Promega) reagents with 100 µM ATP, 0.1 mg/ml 4EBP1 and 5 mM MnCl2 in 25 µl using kinase buffer (40 mM Tris HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml BSA, and 2 mM DTT). Reaction was incubated for 45 minutes at 30°C.
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.