Magnehis protein purification kit

The transcription factor MYC2-like was cloned into pET28a for electrophoretic mobility shift assays (EMSAs). The recombinant vector was transformed into Escherichia coli BL21 competent cells, and the fusion protein was purified with a Magnehis Protein Purification Kit (Promega, Cat. No. V8500). The samples were analyzed by 12% SDS-PAGE and western blots with His tag antibody conjugated to horseradish peroxidase. The oligonucleotides containing three tandem copies of the elements are listed in S1 Table in S1 File and labeled by a Biotin 3’ End DNA Labeling Kit (Thermo, Cat. No. 89818). EMSAs were conducted using a Light Shift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Cat. No. 20148) according to the manufacturer’s instructions.

Nrf2 (subcloned into pGEX5x-1, Amersham) and MafG were expressed as recombinant GST and His-tagged proteins respectively in BL21-CodonPlus (DE3)-RIPL cells (Stratagene). Recombinant MafG was partially purified using the MagneHis Protein Purification kit (Promega) as recommended by the manufacturer. GST-Nrf2 was partially purified with Super-Glu10 resin (Generon). Protein integrity was verified by resolving aliquots on a 4–12% NuPAGE Bis-Tris gel in MES buffer (Invitrogen).