Qproteome Bacterial Protein Prep Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cultures are centrifuged (10,000×g, 10 min, 4°C), pellets are resuspended in 500µL of PBS and centrifuged (14,000×g, 5 min, 4°C). The PBS wash is repeated twice. - Cell pellets are frozen using liquid nitrogen then thawed on ice for ≈15 min.	- Soluble proteins were extracted from the cell pellets using a Qproteome bacterial protein preparation kit according to the manufacturer's protocol..

Upstream tips
- Cultures are centrifuged (10,000×g, 10 min, 4°C), pellets are resuspended in 500µL of PBS and centrifuged (14,000×g, 5 min, 4°C). The PBS wash is repeated twice. - Cell pellets are frozen using liquid nitrogen then thawed on ice for ≈15 min.
Protocol tips
- Soluble proteins were extracted from the cell pellets using a Qproteome bacterial protein preparation kit according to the manufacturer's protocol..

Publication protocol

MudPIT was used to compare the protein expression profile of L. monocytogenes strain EGD-e following adaptation to growth at pH9.0 (±0.2). Replicate 10 mL pH7.3 and 9.0 adapted cultures were prepared, incubated at 37°C, and harvested at late exponential phase (OD600 ≈0.5–0.6; Figure 1) for proteomic analysis. The cultures were centrifuged at 10,000×g for 10 min at 4°C and the supernatant was discarded. The pellets were resuspended in 500 µL of phosphate buffered saline (PBS; pH7.3 and pH9.0±0.2 respectively) and transferred into 1.5 mL Eppendorf Protein Lobind microcentrifuge tubes (Sigma-Aldrich, Castle Hill, NSW, Australia). The tubes were centrifuged at 14,000×g for 5 min at 4°C and the PBS supernatant was discarded. The PBS wash was repeated twice. The cell pellets were frozen using liquid nitrogen then thawed on ice for ≈15 min. Soluble proteins were extracted from the cell pellets using a Qproteome bacterial protein preparation kit (37900; Qiagen Pty. Ltd., Victoria, Australia) and approximate concentrations of the protein extracts was determined using a Pierce BCA Protein Assay kit (ThermoFisher Scientific, Victoria, Australia) according to manufacturer instructions.

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Manufacturer protocol

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