To separate supernatant and OMV fractions, WT, ΔgltA, ΔgltB, and ΔgltK vegetative cells were grown in CYE medium to OD600 0.7. Intact cells were first eliminated by sedimentation at 7830 rpm (10 min, RT). After addition of 1 mM phenylmethylsulfonyl fluoride, supernatants were sedimented at 125,000g (2 hours, 4°C). The resulting pellets (OMV fraction) and supernatants (soluble fractions) were then treated separately. The OMV pellets were washed with TPM, sedimented again at 125,000g (2 hours, 4 °C), and then resuspended directly in 500 μl of 1× Laemmli protein sample buffer. The soluble supernatant fractions were treated with TCA (10% final concentration) for 30 min on ice and then sedimented at 11,000 rpm (1 hour, 4°C). The resulting pellets (precipitated proteins) were washed with 100% acetone, sedimented at 7830 rpm (10 min, 4°C), and dried overnight at RT. Dried pellets were then resuspended in 1.5 ml of TPM, sedimented at 15,000 rpm (30 min, 4°C), and lastly resuspended in 500 μl of 1× Laemmli protein sample buffer.
For isolation of supernatant-alone samples, 10 ml of CYE cultures (inoculated at OD600 0.02) were grown overnight with shaking (220 rpm, 32°C) to OD600 0.6 to 1.0 and then sedimented (5000g, 10 min, 20°C). Supernatants were then sedimented in an ultracentrifuge (Beckman, SW 41 Ti rotor, 120,000g, 75 min, 4°C) to remove any remaining membrane material. Clarified 10 ml of supernatant samples was treated with 1 ml of 100% TCA to precipitate the proteins. Tubes were heated at 65°C for 5 min and then spun in a centrifuge (16,300g, 20 min, RT) to sediment precipitate in 2-ml microtubes. TCA-precipitated pellets were washed with 1 ml of acetone and sedimented (16,300g, 20 min, RT), followed by supernatant aspiration. Protein pellets were left uncapped in the chemical hood overnight to ensure evaporation of acetone. Pellets were resuspended in 500 μl of 2× Laemmli sample buffer lacking reducing agent and then diluted to 1× with ddH2O.
For isolation of supernatant-alone samples, 10 ml of CYE cultures (inoculated at OD600 0.02) were grown overnight with shaking (220 rpm, 32°C) to OD600 0.6 to 1.0 and then sedimented (5000g, 10 min, 20°C). Supernatants were then sedimented in an ultracentrifuge (Beckman, SW 41 Ti rotor, 120,000g, 75 min, 4°C) to remove any remaining membrane material. Clarified 10 ml of supernatant samples was treated with 1 ml of 100% TCA to precipitate the proteins. Tubes were heated at 65°C for 5 min and then spun in a centrifuge (16,300g, 20 min, RT) to sediment precipitate in 2-ml microtubes. TCA-precipitated pellets were washed with 1 ml of acetone and sedimented (16,300g, 20 min, RT), followed by supernatant aspiration. Protein pellets were left uncapped in the chemical hood overnight to ensure evaporation of acetone. Pellets were resuspended in 500 μl of 2× Laemmli sample buffer lacking reducing agent and then diluted to 1× with ddH2O.
