Cuso4 5h2o

The bacterial strain Pseudomonasputida MnB1 [American Type Culture Collection (ATCC) no. 23483] was cultured in the Lept medium (0.5 g/L yeast extract (Ruji, Shanghai, China), 0.5 g/L Casamino Acids (Coolaber, Beijing, China), 5 mM D(+)-glucose (Macklin, Shanghai, China), 10 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.5, GBCBIO, Guangzhou, China), 0.48 mM CaC1₂ (Macklin), 0.83 mM MgSO₄ (Macklin), 3.7 μM FeCl₃ (Macklin), and 1 mL of trace element solution (10 mg/L CuSO₄•5H₂O, 44 mg/L ZnSO₄•7H₂O, 20 mg CoCl₂•6H₂O and 13 mg/L Na₂MoO₄•2H₂O, (Macklin)) containing 1mM MnCl₂ (Aladdin, Shanghai, China) at 30 °C and shaken at 150 rpm for 5 d [48 (link)]. The suspensions were centrifuged at 8000× g for 20 m and the supernatant was discarded, sediments of bacteria and BMO were diluted three times with deionized water by means of centrifugation (20 m at 8000× g), then the mixture of precipitates were collected for freeze-drying to obtain the dried BMO sample.

All chemicals were of analytical grade and were used without further treatment. Phosphate buffer solution (PBS, 0.1 M) was prepared by mixing NaH₂PO₄ and Na₂HPO₄, obtained from Macklin. CuSO₄·5H₂O (99%), NaCl, NaBr, NaI, 4,4’-bipyridine, and ethanol were bought from Macklin. Hydrogen peroxide (H₂O₂, 30 wt%) was purchased from Beijing Chemicals (Beijing, China) and stored in a 4 °C refrigerator. Ultrapure water was used to prepare all aqueous solutions throughout a Millipore system (18.2  MΩ; Millipore Co., USA).

2 mL positive probe storage solution and 2 mL negative probe solution were added to the PC-3 containing medium to achieve a final concentration of 20 µM. The above liquid was incubated at 37 ℃ for 2 h. Then, cells were collected, proteins were prepared by 30% power ultrasound and the protein concentration was regulated by PBS at 1 mg/mL to 2 mg/mL. The samples were grouped and added into 5 mM biotin azide compound of 11.3 µL, 50 mM TCEP (Aladdin, Shanghai, China) of 11.3 µL, 1.7 mM TBTA (Aladdin) of 34 µL and 50 mM CuSO₄·5H₂O (Macklin, Shanghai, China) of 11.3 µL. The protein was incubated at room temperature for 1 h to precipitate. After centrifugation, pre-cooled methanol was added for washing three times and the protein was dissolved. 50 µL streptavidin magnetic beads were added to each group of samples, and the magnetic beads were precipitated and washed three times. The supernatant of samples was discarded and 30 µl PBS (Gibco, CA, USA) and 30 µL 5X protein loading buffer was added to the samples for boiling at 95 ℃ for 10 min. SDS-PAGE electrophoresis was performed and the gel was stained with silver or coomassie bright blue.