Anti mouse horseradish peroxidase labeled goat antibody

The wild‐type PfAspAT ORF was re‐cloned into pASK‐IBA3 and the resulting plasmid‐encoded full‐length PfAspAT‐WT with C‐terminal Strep‐tag. The His₆‐tagged PfAspAT‐Y68A/R257A double mutant was sub‐cloned into pACYC184 vector (NEB) containing the expression cassette of pJC40 to allow co‐expression of the WT and mutant version in E. coli. The co‐expression of Strep‐tagged PfAspAT‐WT and His₆‐tagged PfAspAT‐Y68A/R257A was performed using co‐transformed BLR (DE3) competent cells induced with both IPTG and AHT. The co‐purification was performed via the Strep‐tactin as well as via Ni‐NTA agarose (Qiagen). The co‐purified proteins were visualized by western blot using a monoclonal Strep‐tag II antibody (IBA) or anti‐His antibody (Pierce, USA) and a secondary anti‐mouse horseradish peroxidase‐labeled goat antibody (Bio‐Rad, Germany).

Wild type PfMDH open reading frame was re-cloned into pASK-IBA3 using a modification of the protocol described in [12 (link), 23 (link)] (Table 4). Resulting plasmid encoded full-length PfMDH-WT with C-terminal Strep-tag. Expression of both Strep-tagged PfMDH-WT and His₆-tagged PfMDH-V190W mutant was performed as described above. The lysates were separately clarified by centrifugation, mixed and incubated for 2 hours at 277 K [42 ]. The subsequent co-purification from the mixed lysates was performed via the Strep-tactin as well as via Ni-NTA agarose (IBA Lifesciences, Qiagen).
Co-purified PfMDH-WT and PfMDH-V190W were visualized by western blot using a monoclonal Strep-tag II antibody (IBA) or anti-His antibody (Pierce, USA) and a secondary anti-mouse horseradish peroxidase labeled goat antibody (BioRad, Germany) as described in [42 ] (Fig 6).