Anti histidine his6 horseradish peroxide conjugated antibody from mouse

For purification, cells were harvested 24 h post induction at 13,600 × g at 4 °C, resuspended in phosphate lysis buffer (500 mM NaCl, 25 mM sodium phosphate pH 7.4, 20 mM imidazole) containing 1 mM PMSF, 30 μg/mL lysozyme, and 1 mM EDTA. The cell lysate was frozen at -80 °C overnight. The cells were retrieved and thawed on ice, and 0.1% (v/v) poly (ethyleneimine) (PEI) was added to precipitate nucleic acids followed by sonication for 30 s (3 cycles) at 60 Hz. The sonicated cell lysate was centrifuged for 20 min at 13,600 × g at 4 °C. The supernatant was incubated in nickel-charged nitrilotriacetic acid resin (Ni-NTA) (Thermo Scientific) beads for overnight at 4 °C. Flow through was collected, column was washed with wash buffer A (500 mM NaCl, 25 mM sodium phosphate pH 7.4, 25 mM imidazole) twice and once with wash buffer B (500 mM NaCl, 25 mM sodium phosphate pH 7.4, 80 mM imidazole), and protein was eluted with elution buffer A (500 mM NaCl, 25 mM sodium phosphate pH 7.4, 250 mM imidazole) twice and once with elution buffer B (500 mM NaCl, 25 mM sodium phosphate pH 7.4, 500 mM imidazole). The efficiency of recombinant protein purification was analyzed by 12% SDS-PAGE and western blot using a monoclonal anti-Histidine (His6)-horseradish peroxide-conjugated antibody from mouse (Thermo Fisher Scientific Inc., SA).

The DNA segment encoding PfHsp90 (Plasmodb. Id; PF3D7_0708400) was cloned into pET28a(+) using Xho1 and Nco1 restriction enzymes. pET28a (+) _PfHsp90 plasmid was transformed in E.coli BL21 (DE3). Single colony containing the transformed plasmid was inoculated in 100-mL LB broth (1% w/v sodium chloride, 0.5% w/v yeast, and 1% w/v tryptone) enriched with 50 μg/mL kanamycin and grown at 37 °C for overnight shaking at 200 rpm. The overnight culture was sub-inoculated in 1-L fresh LB broth (1:10) containing 50-μg/ mL kanamycin. The culture was grown at 37 °C until it reaches optical density at 600-nm wavelength (OD600) 0.4-0.6 at 140 rpm shaking incubator (Merck). The protein expression was induced by 0.5 mM IPTG for 24 h at 20 °C. Hourly samples were collected for 5 h post induction and harvested at 13,600 × g for 10 min at 4 °C. The pellet was resuspended in 1X Phosphate Buffer Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 1. 46 mM KH 2 PO 4 ). The expression was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie brilliant blue, and visualized with ChemiDoc™ MP Imaging system (Bio-Rad, USA) and further analyzed by western blot using a monoclonal anti-Histidine (His6)-horseradish peroxideconjugated antibody from mouse (Thermo Fisher Scientific Inc., SA).