N-terminal FLAG-tag(p110alpha), no tag on p85alpha
Description
Complex of N-terminal FLAG-tagged recombinant full-length human p110alpha (GenBank Accession No. NM_006218), with E545K mutation, and recombinant full length, human p85alpha (no tag, GenBank Accession No. NM_181523). Co-expressed in Sf9 cells in a Baculovirus expression system. p110alpha(E545K) MW=125 kDa, p85alpha MW=84 kDa.
Host cell line: Sf9 cells
Specific activity: 800 pmol/min/ug
Unit definition: A unit of p110alpha(E545K)/p85alpha kinase activity is defined as the amount of enzyme required to produce 1 pmol of Phosphoinositol-3, 4,5-Trisphosphate/min at 37°C.
Formulation
25 mM Tris-HCl, pH 8.0, 69 mM NaCl, 1.35 mM KCl, 0.03% Tween-20, 3 mM DTT, 50% glycerol.
Species
Human
Application
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling
Notes
A 25 ul kinase reaction is conducted in a buffer containing 40 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 100 uM ATP, 200 uM PI:PS substrate (BPS catalog #40560) and p110alpha(E545K)/p85alpha enzyme for 15 minutes at 30°C. ATP reduction is detected using ADP-Glo Kinase Assay Platform (Promega Corporation, Madison, WI).
1. Engelman, J.A., et al. Nature Med. 14:1351-1356 (2008) 2. Hafner, C., et al., Proc. Natl. Acad. Sci. USA. 104(33):13450-13454 (2007) 3. Serra, V., et al., Cancer Res.68(19):8022-8030 (2008)