Protein isolation Bacteria - Salmonella enterica

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

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4 years ago

4 years ago by 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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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).
Upstream tips
- For soluble protein preparations from up to 4.5 liters of bacterial cell culture.

- Before use, Native Lysis Buffer must be supplemented with lysozyme and Benzonase® Nuclease.
Downstream tips
- Downstream applications are SDS-PAGE and mass spectrometry.

- Solubilization of inclusion bodies requires high concentrations of denaturants (e.g., 6M Guanidine HCl).
Protocol tips
- Bacteria are centrifuged and pellets are suspended in 1 ml of 1X CelLytic™ B cell lysis reagent (Sigma), containing 0.2 mg/ml lysozyme (RPI Corp.), 50 U/ml Benzonase® Nuclease (Sigma), and 25 μl of Protease Inhibitor Cocktail (Sigma).
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