Irdye 800cw goat anti rabbit igg antibody

For quantitative kinetic and binding assays, pS45-β-catenin was obtained by co-expression with CK1α in E. coli and purified as described above for β-catenin.
pS45-β-catenin could also be obtained by in vitro phosphorylation of β-catenin with purified CK1α. The phosphorylation reaction was performed in a 5 mL volume with 5 μM β-catenin, 1 μM CK1α, and 500 μM ATP in 40 mM HEPES pH 7.4, 50 mM NaCl, 10 mM MgCl₂, 0.05% IGEPAL. The reaction was incubated at 25 °C for 1.5 hours (Figure S1C). Kinetic data obtained with this preparation of pS45-β-catenin support the same conclusions as for pS45-β-catenin obtained from co-expression (Figure S8 & S17, Table S3).
For both co-expressed and in vitro phosphorylated pS45-β-catenin, the extent of pS45 phosphorylation was evaluated by western blotting using a primary anti-phospho-β-Catenin (Ser45) antibody (Cell Signaling Technology #9564) and a secondary IRDye 800CW Goat Anti-Rabbit IgG antibody (Li-Cor #926-32211).