5% of NaCl. As the original NB contains 0.5% NaCl, NBs with 0% and 2.5% NaCl were termed NB (0.5) and NB (3.0), respectively. After cultivation at 37°C for 24  hrs with shaking (140  r.p.m.), the culture supernatant was separated from the cells by centrifugation. Proteins in the culture supernatant of A. sobria were

precipitated by treatment with TCA solution, which was added to 1.0  mL culture supernatant to reach 10% concentration. The mixture was left for 30  min at room temperature and the precipitates yielded collected by centrifugation. After rinsing with ethanol, the precipitates were solubilized with 100 μL loading solution for SDS-PAGE and a portion (15 μL) of the sample loaded onto a lane of SDS-polyacrylamide gel. The concentration of acrylamide in the gel used was 15%. A portion of overnight preculture of A. sobria 288 (asp−, amp−) (20  mL) was inoculated into GSK-3 inhibitor review selleck kinase inhibitor 2 liters of NB (0.5). After cultivation at 37°C for 24  hrs with shaking (140  r.p.m.), the culture supernatant was separated from the cells by centrifugation (12,000  g for 10  min) at 4°C. The culture supernatant was salted out with 30% saturated ammonium sulfate

and the insoluble materials removed by centrifugation. Ammonium sulfate was added to the supernatant to reach 50% saturated ammonium sulfate. The insoluble materials yielded were collected by centrifugation and dissolved in 10  mL of 10 mM phosphate buffer (pH 7.4). The samples were dialyzed against the buffer. The prepared samples were designated the crude samples. One milliliter of the crude sample was loaded onto a hydroxyapatite column (CHT10-I) (Bio Rad, Hercules, CA, USA) equilibrated with 10  mM phosphate

buffer (pH 7.4). Non-adsorbed materials were washed out with 10  mM phosphate buffer, and materials adsorbed to the column eluted with a linear gradient of 10 to 300  mM phosphate buffer (pH 7.4). The fractions containing the target protein were detected by SDS-PAGE. The fractions containing the target GNA12 protein were collected and concentrated by an Amicon ultra-15 centrifugal filter tube (Millipore, Billerica, MA, USA). A portion of the concentrated sample (250 μL) was loaded onto a Superdex 75 column (column size, 10 mm ×  300  mm; GE Healthcare UK, Buckinghamshire, UK) equilibrated with 50  mM phosphate buffer (pH 7.4) containing 150  mM NaCl. After loading the sample, the column was eluted with the buffer used for equilibration. The fractions containing the target protein obtained by column chromatography using Superdex 75 were collected and concentrated by an Amicon ultra-15 centrifugal filter tube. The concentrated sample was separated by SDS-PAGE. Proteins on the gel were transferred to a PVDF membrane on trans-blot apparatus for 30  min at 160  mA at room temperature, and the membrane stained with Coomassie brilliant blue.