Protein isolation Bacteria - Mycobacterium smegmatis

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
- 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.
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