Trichloroacetic acid

Protein isolation Bacteria - Clostridium difficile

Experiment
Protein isolation Bacteria - Clostridium difficile
Product
Trichloroacetic acid from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

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.

Publication protocol

C. difficile strains were grown at 37°C in TY medium to stationary phase (24 h). Cells were removed by centrifugation at 8,000 × g for 10 min. The supernatants were retained and sterilized by filtration through a 0.22-μm-pore-size Millex GP filter (Millipore, Billerica, MA). Next, 20 ml of culture supernatants was precipitated by the addition of 100% (wt/vol) trichloroacetic acid (TCA) at −20°C to a final TCA concentration of 10%. Solutions were incubated on ice for 45 min before centrifugation at 4,500 × g for 45 min at 4°C. The supernatants were discarded, and 3 ml of ice-cold acetone was added to the pellet. The sample was then held on ice for 15 min before centrifugation at 4,500 × g for 45 min. The supernatant was discarded, and the pellet was air dried before resuspension in 130 μl of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 1% DTT, 0.5% ASB-14 detergent in Milli-Q water). Protein concentrations were determined using a Bradford protein assay (Bio-Rad, Mississauga, ON, Canada). A standard curve was generated using a bovine serum albumin (BSA) standard kit (Bio-Rad, Mississauga, ON, Canada).

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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

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Trichloroacetic acid below.

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