| Assay Method Information | |
| | Biochemical Assay |
| Description: | Full-length wild-type β-catenin (residues 1-781), full-length β-catenin D145 Å, or full-length β-catenin E155A were cloned into a pET-28b vector carrying a C-terminal 6× histidine (Novagen), and transformed into E. coli BL21 DE3 (Novagen). Cells were cultured in LB medium with 50 μg/mL kanamycin until the OD600 was approximately 0.8, and then protein expression was induced with 400 μM of IPTG at 20° C. overnight. Cells were lysed by sonication. The proteins were purified by three steps of chromatography, including Ni-NTA affinity chromatography (30210, Qiagen), HiTrap Q HP anion exchange chromatography (17-1154-01, GE Healthcare Life Science) and size-exclusion chromatography with a HiLoad 26/600 Superdex 200 pg column (28-9893-36, GE Healthcare Life Science) using an AKTA Pure FPLC (GE Healthcare Life Science) system. Protein was eluted in a buffer containing 20 mM of Tris (pH 8.5), 100 mM NaCl, and 2 mM DTT. The purity of β-catenin was greater than 95% as determined by SDS-PAGE gel analyses. Thermal-shift assays were performed on an CFX96 Real Time System (Bio-Rad) to monitor protein stability and detect protein aggregation. Protein unfolding was evaluated through measurement of the fluorescence changes of fluorescent dye Sypro Orange when interacting with β-catenin proteins. A temperature increment of 1°/min was applied. All proteins were stable and no aggregation was observed under storage or assay conditions. Proteins were aliquoted and stored at −80° C.BCL9 Peptide Synthesis and Purification. Human BCL9 (residues 350-375), N-terminally biotinylated human BCL9 (residues 350-375) and N-terminally biotinylated human E-cadherin (residues 824-877) were synthesized by InnoPep Inc. (San Diego, CA, www.innopep.com). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |