Anti 6x his tag monoclonal antibody

To further increase the protein expression of recombinant TP4 in P. pastoris, the codon-optimized TP4 gene was designed to match P. pastoris expression by artificial gene synthesis from Omics Bio (Taipei, Taiwan). The nucleotide sequence products were confirmed by sequencing using primers, and then the identified plasmid was digested with EcoRI and XbaI. After enzyme digestion, the insert was ligated into the pPICZαA vector. The pPICZαA plasmid ligated with TP4 was digested with PmeI to linearize the vector and then transformed into P. pastoris X-33 by electroporation. The P. pastoris X-33 strain integrated with recombinant pPICZαA-TP4 DNA was grown in YPD broth at 30°C with 250 rpm agitation for 16 h. The resulting culture was inoculated into 30 mL of BMGY medium and incubated for 24 h at 30°C. The P. pastoris culture was then harvested by 10 min of centrifugation at 3,000 xg. The cell pellet was resuspended in 20 mM phosphate buffer, sonicated (400 cycles with 3 sec on/3 sec off) on ice by using a Sonicator 3000 system (HEAT Systems Inc., Farmingdale, NY) and then centrifuged at 5,000 xg for 20 min at 4°C. The recombinant protein in the pellet and supernatant was detected by SDS-PAGE and Western blotting using an anti-6x-His tag monoclonal antibody (Invitrogen, MA1-21315).

HEK293 cells were seeded in six-well plates of 5x10⁵ cells per well. After the cells are completely attached, HEK293 cells were infected with VV-Ctrl-BiTE or VV-EpCAM BiTE at the MOI of 1. After 48 hours, the supernatants were harvested and centrifuged to remove cells or cellular debris. The primary antibody was anti-6x-His tag monoclonal antibody (Cat# MA1-21315, Invitrogen). The secondary antibody was Goat anti-Mouse IgG (H+L) (Cat# A16068, Invitrogen).