Runblue

Intracellular localization of proteins was further analyzed by fractionated cell disruption. Here, cells (from 1 mL culture) were harvested either 1 h (for immunoblot analysis) or 3 h (for Instant Blue staining) after induction of protein expression. The cell pellet was resuspended in 50 µL resuspension buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl; 10 mM EDTA, 1 × HP-protease inhibitor mix (Serva)) and cells were broken by repeated cycles of freeze and thaw. Afterwards, cells were adjusted to a final OD₆₀₀ of 5 in resuspension buffer supplemented with benzonase (≥500 units; Sigma Aldrich). After 20 min incubation on ice, cells were spun down for 1 min at 500 × g to remove cell debris. The supernatant containing all soluble and insoluble proteins was transferred to a fresh reaction tube. An aliquot of the supernatant was taken, representing the total protein fraction (total). The remaining cell lysate was spun down for 15 min at 20,000 x g and the supernatant containing all soluble proteins was transferred into a new reaction tube (sol). The isolated fractions were separated on SDS-PAGE (RunBlue 4–20 %, Expedeon; NuPAGE^®Bis-Tris gel 4–12%, Invitrogen) and analyzed by Instant Blue staining (Expedeon) and quantitative immunoblotting using an anti-His antibody (Novagen).

SDS-PAGE was performed according to the method of Laemmli (1970 (link)), using a separation gel of 12% acrylamide. Phage lysate (7–14 μl) were dissolved in (3–6 μl) SDS-PAGE sample buffer (containing 2-mercaptoethanol), mixture was then heated at 100°C for 5 min. Samples were then inoculated into SDS-PAGE gel and a protein ladder (NEB™) of size ranging from 6.5 to 212 kDa, was used as protein size marker. After electrophoresis the acrylamide gel was submerged in 20 ml of “instant blue” (a coomassie based blue staining solution) obtained from RunBlue™, Expedeon, for 1 h on an orbital shaker and then placed in the fridge overnight. The resulting protein band sizes were determined using GelCompar II software.