Protein isolation Bacteria - Listeria monocytogenes

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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Protocol tips
- Bacterial pellets are recovered after centrifugation (10,000rpm, 10 min), washed twice in PBS, and resuspended in 100 µl of B-PER II bacterial protein extraction reagent following the manufacturer's protocol.
Downstream tips
- Culture supernatants are precipitated in 60% ammonium sulfate or trichloroacetic acid at 4°C overnight. After centrifugation, pellets are washed twice with 5ml cold acetone, dried, and resuspended in 200 µl of 2× Laemmli buffer.
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..
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