Protein Extraction and Western Blot Analysis in L. lactis

To extract proteins from L. lactis cells, 50 ml of cell culture was centrifuged for 12 min at 1700g at 4 °C. The pellet was resuspended in 25 ml of ice-cold washing buffer (300 mM NaCl, 10% glycerol, 50 mM Tris-HCl pH 8.0). Cells were pelleted identically and resuspended in 1.5 ml of lysis buffer (300 mM NaCl, 10% glycerol, 1 mM PMSF, 1/2000 protease inhibitor cocktail, 2 mg/ml lysozyme, 50 mM Tris-HCl pH 8.0). Eight hundred microliters of cell suspension were mixed with 800 mg of glass beads (0.17–0.18 mm diameter), and cell lysis was performed with Precellys apparatus, 5 × 30 s at 5000 rpm. Centrifugation was performed for 12 min at 5000 rpm at 4 °C to remove cell debris, and 500 μl of supernatant was collected for further protein quantification, SDS-PAGE, and Western blotting.
For Western blotting, 20 μg of proteins were separated on SDS-PAGE gels, and Western blotting was carried out as previously described (19 (link)). The primary rabbit antibodies against Gdt1p were previously produced in our lab (9 (link)) and antibodies for sfpHluorin detection were purchased from Chromotek (PABG1, 1:1000 dilution). Horseradish peroxidase–coupled anti-rabbit secondary IgG antibodies and Lumi-Light Western Blotting Substrate (Roche Diagnostics) were used, and chemiluminescence was captured using an Amersham Imager 600 (GE Healthcare) with automatic exposure time for high dynamic range.