Protein isolation Bacteria - Clostridium difficile

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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5 years ago

5 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
- Cells are removed by centrifugation at 8,000 × g for 10 min. The supernatants are retained and sterilized by filtration through a 0.22-μm-pore-size Millex GP filter (Millipore, Billerica, MA).
Protocol tips
- 20 ml of culture supernatants are precipitated by the addition of 100% (wt/vol) trichloroacetic acid (TCA) at −20°C to a final TCA concentration of 10% (incubated on ice for 45 min before centrifugation at 4,500 × g for 45 min at 4°C). 3 ml of ice-cold acetone is added to the pellet (15min before centrifugation at 4,500 × g for 45 min). The pellet is air dried before resuspension in 130 μl of rehydration buffer.
Protocol tips
Spore samples were harvested at 72 h, resuspended to 1 × 107 spores/100 μl in 1× Laemmli loading buffer, and heated to 95°C for 5 min. Spore extracts were separated on 3 to 8% NuPAGE Novex Tris-acetate minigels and blotted onto polyvinylidene difluoride (PVDF).
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