Pierce c myc peptide

To generate and purify Flag-TAZ/Myc-USP7-WT recombined proteins from mammal cells, HEK293T cells were transfected with the indicate plasmid and cultured in SFM4Transfx-293 without L-Glutamine culture medium (SH30860.02, Thermo) for another 4 days. Cells were harvested and the recombined proteins were purified by using Pierce™ anti- DYKDDDDK Magnetic Agarose (A36797, Thermo)/ Pierce™ Anti-c-Myc Agarose (20168, Thermo) and then eluted with Pierce™ 3×DYKDDDDK peptides (A36805, Thermo)/Pierce™ c-Myc Peptide (20170, Thermo). The purified proteins were verified by SDS-PAGE.
The purified Flag-TAZ and Myc-USP7-WT were mixed with equimolar, incubated (supplemented with protease inhibitor mixture and Pierce™ anti- DYKDDDDK Magnetic Agarose) at 4 °C overnight, and followed by 5-time IP lysis buffer washing step. The magnetic agarose was resuspended and routinely subjected to SDS-PAGE process.

MYC-TRAF4 or Flag-HER2 expressing plasmid was transiently transfected into HEK293T cells. Expressed protein was then purified using anti-MYC beads or anti-FLAG beads and eluted using 0.5 mg/mL Pierce c-Myc-Peptide (20170, Thermo Fisher Scientific) or 3X FLAG peptide (A36805, Thermo Fisher Scientific). In vitro ubiquitination assays were performed using ubiquitination kit (BML-UW9920-0001, Enzo Life Sciences, Farmingdale, New York, USA) following the manufacturer’s instructions. Briefly, the reaction was set up in a total of 25 μL volume with the following components: 2.5 μM biotinylated ubiquitin, 100 nM E1 enzyme, 1 μM E2 enzymes (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, UbcH13/Mms2), 100 ng active human SMURF2 protein and purified TARF4 or HER2 protein. The mixture was incubated at 37 °C for 2 h and quenched by adding 2× non-reducing gel loading buffer before being analyzed by western blot analysis.