This procedure is suitable for precipitating peptides from organic acids and is used routinely for concentrating synthetic peptides from the trifluoroacetic acid cleavage cocktail following 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. It can be used to desalt peptides in nonvolatile buffers but is not recommended for removing detergents like SDS. Take appropriate safety precautions when using this technique; ether is highly flammable, and a refrigerated centrifuge and fume hood are recommended. Practice the precipitation procedure with 5- to 25- |g amounts of a standard peptide before attempting use with a precious sample. Maintain cold solution temperatures for best results.
Additional Materials (also see Basic Protocol)
75% trifluoroacetic acid (Sequenal grade TFA, Pierce) Ether, ice-cold
6 x 50-mm tube Centrifuge, 4°C
CAUTION: Ether is highly flammable; work with a fume hood.
1. Vacuum dry an aliquot of the peptide preparation in a 6 x 50-mm tube. Redissolve the peptide in 50 to 200 |l of 75% TFA and cool to 4°C on ice.
Alternatively, the peptide preparation can be diluted with 100% TFA to a final concentration of 75% TFA and cooled to 4°C. Keep final sample volumes small (e.g., ~200 ul).
2. Add 3 vol ice-cold ether, mix, and incubate 15 to 30 min on ice or in a -20°C freezer.
Flocculent peptide is usually visible above 20 ug but increasingly difficult to see below 20 ug.
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