Roti fluoro pvdf

The SDS-PAGE was performed as described by Laemmli^{32 (link)}. The separation of protein samples was carried out with 12% SDS polyacrylamide gel. The SDS polyacrylamide gels were stained using Coomassie brilliant blue R250 solution for analysis as described earlier^{33 (link)} for visual observation. For immunoblotting the protein samples from the SDS-PAGE were transferred onto a polyvinylidene fluoride (PVDF) membrane (Roti®-Fluoro PVDF, pore size 0.2 μM, Carl Roth, Germany) using Trans-Blot SD Semi Dry Transfer Cell (Bio-Rad, USA). Subsequently, the membrane was soaked in a blocking buffer, 5% nonfat dry milk in tris-buffered saline (W/V) with 0.5% tween 20 (V/V) (TBS-T) for 1 h at room temperature with a constant shaking (20 cycles/min). Afterwards, the membrane was probed with the respective primary antibody in a blocking buffer (30 cycles/min), followed by three washes with TBS-T buffer for 10 min and incubated with blocking buffer containing the respective secondary antibody. After three washes with TBS-T buffer, blots were visualized by a commercially available enhanced chemiluminescence (ECL) detection kit (PerkinElmer, USA) and the images were acquired by FUSION FX7™ Advance Chemiluminescence System (Peqlab, Germany).

The presence of GvpE in transformants harboring derivatives of pLacZJB18+E was determined by Western analysis. Transformants were grown in 50 ml cultures to OD₆₀₀ 1.2, harvested by centrifugation (2,000 ×g, 45 min, 4°C) and the sediment was resuspended in 2–3 ml lysis buffer (2.5 M KCl, 50 mM MgCl₂, 1 mM EDTA, 5% [v/v] glycerol, 50 mM Tris-HCl pH 8.0). The cells were disrupted by sonication on ice (2 × 5 min, Branson sonifier 250, 3 mm disruptor horn) and the suspension centrifuged at 2,000 ×g for 45 min at 4°C. To remove the high amount of salt the suspension was dialyzed against 10 mM Tris-HCl pH 7.2 for 12 h. After dialysis, 20 μg of the total protein were separated by SDS-PAGE (Schägger and von Jagow, 1987 (link)) and transferred to a PVDF membrane (Roti^®-Fluoro PVDF, Carl Roth) using the PerfectBlue^TM “Semi-Dry”-Blotter. The membrane was subsequently incubated for 1 h at 37°C, reactivated in 100% methanol, washed for 4 min with PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 20 mM KH₂PO₄, pH 7.4) and blocked for 1 h with Odyssey Blocking Buffer (LI-COR). After incubation with the GvpE antiserum in Odyssey Blocking Buffer overnight, the membrane was washed four times for 5 min with PBS + 0.1% (v/v) Tween^® 20 and incubated with the secondary antibody IRDye 800CW (LI-COR) coupled with a fluorophore detectable at 800 nm. The detection was done with the Odyssey Fc Imager (LI-COR).