Peptide Sample Preparation by Ether Precipitation

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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Responses

  • kristian boehm
    What does ether doin protein sample preparation?
    3 years ago

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