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

Protein isolation Bacteria - Salmonella enterica

Experiment
Protein isolation Bacteria - Salmonella enterica
Product
B-PER™ II Bacterial Protein Extraction Reagent (2X) from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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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Discussion

Discussion

5 years ago

Author: Hollie Fowler United Kingdom

How can I deal with my pellet being too viscous?

I am using 8M Urea to lyse my cells but after centrifugation my pellet is very viscous. This causes me trouble when I try to collect the supernatant. Is there any way to avoid it and do you think it will have an effect on the concentration of my protein?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein isolation Bacteria - Salmonella enterica using B-PER™ II Bacterial Protein Extraction Reagent (2X) from Thermo Fisher Scientific.

Paper title
Engineering formation of multiple recombinant Eut protein nanocompartments in
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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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