Ja 20 rotor

Bacteria were initially grown in the 0.3×Marine Broth 2216 medium into exponential phase. Cells were harvested by centrifugation (8000 rpm, 10 min, JA 20 rotor, Beckman). The pellets were washed twice with 0.85% (w/v) NaCl solution, and re-suspended in distilled water. The suspension was inoculated into a nitrogen-free agar plate containing 19.45 g NaCl, 8.8 g MgCl₂, 3.24 g Na₂SO₄, 1.8 g CaCl₂, 0.55 g KCl, 0.16 g NaHCO₃, 0.1 g Ferric citrate, 0.08 g KBr, 0.034 g SrCl₂, 0.022 g H₃BO₃, 8.0 mg Na₂HPO₄, 4.0 mg Na₂SiO₃, 2.4 mg NaF per liter (pH 7.4). Survived bacteria were passaged at least 20 times on the agar plate. The strains Azospirillum lipoferum Sp59^T and Escherichia coli DH5α were included as positive and negative control, respectively.

Cell pellets were washed twice with 20 mM HEPES, 100 mM NH₄Cl, pH7.4; and dissolved in lysis buffer (20 mM HEPES, 100 mM NH₄Cl, 10.5 mM Mg Acetate, 0.5 mM EDTA, 5 mM 2-mercaptoethanol, 3 mM phenylmethylsulfonyl fluoride (PMSF), and Roche cOmplete protease inhibitor tablets). The cells were lysed using Emulsifier EmulsiFlex-C3 at 25,000 PSI for 15 cycles. The lysate was collected and centrifuged at 15,000 RPM for 45 min in a Beckman Coulter JA-20 rotor. The protein concentration was determined via Bradford assay using BSA as a standard. The cell lysate was treated with 1 µg/µL of RNase A and 0.75 µg/µg of DNase for 30 min at 30 °C to remove the DNA and RNA. Approximately, 500 µg of proteins were amidinated with SMTA by following the protocol described above. Excess SMTA was removed by using an Amicon 3kDa MWCO centrifugal filter. Proteins were separated and fractionated by using SCX and C4 reverse-phase trapping columns, as described above. The protein solution was digested, and the resulting peptides were analyzed on a high-resolution Thermo Orbitrap XL and Fusion Lumos mass spectrometer.

The media containing btBDNF were harvested and plates were washed using a washing buffer (30 mM phosphate buffer (pH 8.0), 500 mM NaCl, 20 mM imidazole and a cocktail of protease inhibitors) (Sigma-Aldrich). The media were then incubated on ice for 15 min, centrifuged at 18,000 rpm for 30 min using a Beckman JA-20 rotor and the supernatant was collected. Ni-NTA resins (Qiagen, Hilden, Germany) were rinsed with the washing buffer and added to the collected supernatant at a concentration of 0.3 mL Ni-NTA resins to 100 mL media and incubated overnight on a shaker at 4 °C. The media/Ni-NTA slurry was loaded onto a column and the captured Ni-NTA resins were washed with 10 mL wash buffer and eluted with the elution buffer (30 mM phosphate buffer, pH 8.0, 500 mM NaCl, 300 mM imidazole, protease inhibitors). The purity and concentration of BDNF was assessed by SDS-PAGE using a fast silver staining kit (G-Biosciences, St Louis, MO, USA). Known quantities of BDNF and BSA were used as standards. To precipitate proteins with trichloroacetic acid (TCA), 100% ice-cold TCA was added to the supernatant at a final concentration of 5–7% and incubated on ice for 20 min. The sample was then centrifuged for 20 min at 14,000 rpm. The pellet was washed three times with acetone, air-dried and heated in an SDS loading buffer.