Thick blotting paper

We transferred 10.0 mL of BY-2 cells from a continuous shake flask suspension culture at a density of ~200 g L⁻¹ to a 10-mL syringe (Braun, Melsungen, Germany) equipped with a custom-made filter layer prepared from thick blotting paper (Bio-Rad, Hercules, USA). Residual medium was removed by applying a pressure of ~ 0.2 MPa and the preparative PCP (pPCP) was infiltrated with 6.0 mL R. radiobacter suspension prepared as described above and incubated for 1 h at 22°C. The infiltration suspension was removed and the pPCPs were incubated for 4 days at 26°C and a relative humidity of ~ 55%. Samples were extracted by sonication using a SonoPuls device equipped with a SonoPuls MS73 titanium alloy microtip (Bandelin, Berlin, Germany) at 40 W for 2 × 30 s with a 0.9-s interval. Extracts were clarified by two rounds of centrifugation (4200 × g, 10 min, 10°C) and the supernatant was passed through a hydrophilic 0.2-µm MiniSart syringe sterile filter (Sartorius, Göttingen, Germany) before the chromatography step.