DNA corresponding to deletion constructs of PFA0660w and PfHsp70-x were PCR amplified, and cloned in pET-28a(+) bacterial expression vector before expression in E. coli BL21 (DE3) cells. Recombinant proteins were purified using Ni-NTA affinity chromatography followed by gel permeation chromatography. Gel filtration chromatography was performed using AKTA prime plus on Superdex 200 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA). Purified PFA0660w-S was used for raising polyclonal antibodies in rabbits commercially (Merck, Bangalore, India).
To confirm protein identity, purified recombinant protein constructs of PFA0660w and PfHsp70-x were resolved on SDS-PAGE and transferred on NC membrane. NC membrane was blocked overnight at 4 °C in 5% BSA. Following washing, the blot was probed with horseradish peroxidise (HRP) linked monoclonal anti-hexahistidine antibody (1:2000; Sigma) for 2 hours, and developed with diamino benzidine/H2O2 substrate (Sigma).
Recombinant ATS domain of PF08_0141 was purified for experiments as previously described36 ,37 (link). Briefly, protein was purified using Ni-NTA affinity chromatography followed by Q-Sepharose anion exchange chromatography (GE Healthcare) and then size exclusion on Superdex 200 10/300 GL column (GE Healthcare).
To confirm protein identity, purified recombinant protein constructs of PFA0660w and PfHsp70-x were resolved on SDS-PAGE and transferred on NC membrane. NC membrane was blocked overnight at 4 °C in 5% BSA. Following washing, the blot was probed with horseradish peroxidise (HRP) linked monoclonal anti-hexahistidine antibody (1:2000; Sigma) for 2 hours, and developed with diamino benzidine/H2O2 substrate (Sigma).
Recombinant ATS domain of PF08_0141 was purified for experiments as previously described36 ,37 (link). Briefly, protein was purified using Ni-NTA affinity chromatography followed by Q-Sepharose anion exchange chromatography (GE Healthcare) and then size exclusion on Superdex 200 10/300 GL column (GE Healthcare).
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