Qproteome Bacterial Protein Prep Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- The liquid medium is aspirated from the Petri dishes. A proteinase inhibitor cocktail (Complete; Roche) is added to the Petri dishes and the dishes are frozen at −80°C.	- Qproteome Bacterial Protein Prep kit (Qiagen) is added to each Petri dish. The whole content is transferred into a glass test tube and vortexed (10×30s cycles with 1 min breaks on ice). The beads and cell debris are removed by centrifugation (5 min at 5000g at 4°C) and are rewashed with lysis buffer. - The proteins precipitation and resuspension protocol is the one provided by the publication.

Upstream tips
- The liquid medium is aspirated from the Petri dishes. A proteinase inhibitor cocktail (Complete; Roche) is added to the Petri dishes and the dishes are frozen at −80°C.
Protocol tips
- Qproteome Bacterial Protein Prep kit (Qiagen) is added to each Petri dish. The whole content is transferred into a glass test tube and vortexed (10×30s cycles with 1 min breaks on ice). The beads and cell debris are removed by centrifugation (5 min at 5000g at 4°C) and are rewashed with lysis buffer. - The proteins precipitation and resuspension protocol is the one provided by the publication.

Publication protocol

The liquid medium was aspirated from the Petri dishes, so that the biofilm pellicle dropped onto the glass beads. A proteinase inhibitor cocktail (Complete; Roche) was added to the Petri dishes, and the dishes were then frozen at −80 °C. Prior to protein isolation, the Qproteome Bacterial Protein Prep kit (Qiagen) was added to each Petri dish. The whole content of the Petri dish, including the glass beads, was transferred into a glass test tube and vortexed for 10× 30 s cycles with 1 min breaks on ice. The beads and cell debris were removed by centrifugation (5 min at 5000 g at 4 °C) and were rewashed with lysis buffer. The proteins were precipitated with 6 % TCA (w/v), incubated on ice for 1 h and centrifuged at 10 000 g at 4 °C for 15 min. The pellets were washed with ice-cold acetone and kept overnight in acetone at −20 °C. The samples were centrifuged (15 min at 10 000 g at 4 °C), and the protein pellets were resuspended in phosphate buffer and treated with a 2-D Clean-Up kit (GE Healthcare). The pellets were finally resuspended in sample buffer containing 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 1 % pharmalyte 3–10, 65 mM DTT and bromophenol blue. The protein concentrations were measured using a 2-D Quant kit (GE Healthcare).

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Manufacturer protocol

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