B-PER™ II Bacterial Protein Extraction Reagent (2X)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cultures are harvested by centrifugation (3000 g, 30 mins) and the cell pellet is washed in Buffer A (50 mM Tris pH 8.0, 500 mM KCl, 12.5 mM MgCl2, 1.5% 1,2 propanediol or ethanolamine-HCl).	- Pellets are resuspended in 10 mL Buffer A and cells are lysed by addition of B-PER® II Bacterial Protein Extraction Reagent (60% v/v) (ThermoScientific). - B-PER® II Bacterial Protein Extraction Reagent is supplemented with lysozyme (0.25 mg mL−1), DTT (1 mM), PMSF (0.1 mM) and DnaseI (2 U). - After 30 minutes incubation at RT, cell debris and supernatant are separated twice by centrifugation (9700 g, 5 mins).	- If you want to isolate bacterial microcompartments: BMCs are pelleted from the resulting supernatant by centrifugation (43000 g, 30 mins). The pellet is resuspended in 500–1000 μL Buffer B (50 mM Tris pH 8.0, 50 mM KCl, 5 mM MgCl2, 1% 1,2 propanediol or ethanolamine-HCl).

Upstream tips
- Cultures are harvested by centrifugation (3000 g, 30 mins) and the cell pellet is washed in Buffer A (50 mM Tris pH 8.0, 500 mM KCl, 12.5 mM MgCl2, 1.5% 1,2 propanediol or ethanolamine-HCl).
Protocol tips
- Pellets are resuspended in 10 mL Buffer A and cells are lysed by addition of B-PER® II Bacterial Protein Extraction Reagent (60% v/v) (ThermoScientific). - B-PER® II Bacterial Protein Extraction Reagent is supplemented with lysozyme (0.25 mg mL−1), DTT (1 mM), PMSF (0.1 mM) and DnaseI (2 U). - After 30 minutes incubation at RT, cell debris and supernatant are separated twice by centrifugation (9700 g, 5 mins).
Downstream tips
- If you want to isolate bacterial microcompartments: BMCs are pelleted from the resulting supernatant by centrifugation (43000 g, 30 mins). The pellet is resuspended in 500–1000 μL Buffer B (50 mM Tris pH 8.0, 50 mM KCl, 5 mM MgCl2, 1% 1,2 propanediol or ethanolamine-HCl).

Publication protocol

Overnight cultures were harvested by centrifugation (3000 g, 30 mins) and the cell pellet was washed in Buffer A (50 mM Tris pH 8.0, 500 mM KCl, 12.5 mM MgCl2, 1.5% 1,2 propanediol or ethanolamine-HCl). Pellets (1 g wet weight) were resuspended in 10 mL Buffer A and cells were lysed by addition of B-PER® II Bacterial Protein Extraction Reagent (60% v/v) (ThermoScientific), supplemented with lysozyme (0.25 mg mL−1), DTT (1 mM), PMSF (0.1 mM) and DnaseI (2 U). After 30 minutes incubation at room temperature, cell debris and supernatant were separated twice by centrifugation (9700 g, 5 mins). BMCs were pelleted from the resulting supernatant by centrifugation (43000 g, 30 mins). The small, glassy pellet was resuspended in 500–1000 μL Buffer B (50 mM Tris pH 8.0, 50 mM KCl, 5 mM MgCl2, 1% 1,2 propanediol or ethanolamine-HCl).

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for B-PER™ II Bacterial Protein Extraction Reagent (2X) below.

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