Supersignal west dura extended duration hrp substrate

Strains were grown to saturation (overnight for C. crescentus and A. excentricus, usually 48 hours for A. biprosthecum). OD600 was determined and cells were collected at a normalized density of OD600 = 1/1mL. 1 mL of each normalized culture was pelleted, resuspended in 100 μL water, and prepared for analysis using standard procedures using SDS-PAGE, transfer, and western blotting. 10 μL of each sample was loaded onto Any kD Mini-PROTEAN TGX Precast Protein Gels (BioRad). The JL-8 monoclonal GFP antibody (Clontech) was used as the primary antibody and Goat Anti-mouse HRP (Pierce) was used for the secondary antibody. Transferred blots were visualized with SuperSignal West Dura Extended Duration HRP substrate (ThermoFisher Scientific) using a Bio-Rad Chemidoc.

After growing for 4 h at 20 °C, OD600 was determined and cells were collected at a normalized density of OD600 = 1/1mL. One mL of the normalized sample was pelleted at 4000g for 15 min and the pellet resuspended in 250 μL 20% sucrose, 1 mM EDTA, 30 mM TRIS pH 8 at room temperature. The sample was mixed gently by rotation at room temperature for 10 minutes before being spun down at 13,000g for 10 minutes. The supernatant was carefully removed and the pellet rapidly suspended in 250 μL ice-cold pure water. The sample was mixed gently by rotation at 4°C for 10 minutes before being spun down at 13,000g at 4°C. The supernatant (periplasmic fraction) and pellet (cell fraction) were then separated and prepared for analysis using standard procedures using SDS-PAGE, transfer, and western blotting. Blots were incubated with His-Probe Antibody (H-3) sc-8136 HRP (Santa Cruz Biotechnology) and visualized with SuperSignal West Dura Extended Duration HRP substrate (ThermoFisher Scientific) using a Bio-Rad Chemidoc.