Protein pellets were dried and stored at −80 °C or immediately dissolved using protein extraction buffer composed of 8 M urea, 50 mM triethylammonium bicarbonate, pH 8.5, for 1 h at 6 °C under constant shaking.
P. cutis was cultured in 250 mL of YM broth (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, Difco) for 3 days at 30 °C under constant shaking (150 rpm), and the cells were collected by centrifugation.
The cultures were grown under constant shaking at 37°C until the optical density at 600 nm reached 0.8.
A 100 μM monomeric E46K solution in 10 mM Tris-HCl, 50 mM NaCl, pH 7.4 was incubated at 70°C in Eppendorf tubes under constant shaking.
Finally, 100 μl thiolated mAb was added in 100 μl MBS-activated NPs for 30 min at 25 °C under constant shaking for preparation of PLGA-PEG immunonanoparticles. .
Bacterial cells of Pseudomonas aeruginosa and Staphylococcus aureus were cultured in Mueller-Hinton (MH) broth to its log growth-phase at 37°C under constant shaking at 180 rpm, respectively.
Cultures were routinely set up in TB medium (500 ml volume), incubated at 37°C under constant shaking (180 rpm), and expression was induced by adding IPTG (final conc. 1 mM).
Prior to the addition of copper compounds, Escherichia coli (NCTC11100) and Staphylococcus aureus RN4220 [29] were grown overnight at 30 °C under constant shaking in an Infors HT Minitron incubator at 80 rpm.
Each bacterial isolate was cultivated in 6 mL KB at 28°C for 24 h in 15 mL cylindrical plastic microcosms under constant shaking at 130 rpm and then plated on KB-agar at 28°C and grown for 48 h.
For thiolation of anti-EGFR antibody, 5.7 mg of 2-iminothiolane (Traut's reagent) was dissolved in 5 ml PBS (pH 7.4) and 100 μl anti-HER-2 mAb was incubated with 4.2 μl of 2-iminothiolane for 2 h at 20 °C under constant shaking.
This mixture was incubated for 2 h under constant shaking at RT.
