Acidified acetone/methanol precipitation method experiment

Summary

Another useful alternative to TCA precipitation is the use of an acidified acetone-methanol mixture. Acidification helps to remove SDS from the sample by dissolving the detergent in the organic phase while the protein produces a precipitate. both acetone and methanol can be dissolved in water in any ratio, so there is no phase separation. This experiment is from the Protein Purification and Characterization Lab Guide by Houzhu Zhu.

Operation method

Acidified acetone/methanol precipitation method experiment

Materials and Instruments

Hydrochloric acid Acetone Methanol

Move

makings

Hydrochloric acid (high purity; e.g., J,T-Baker's Ultrex grade)

Acetone (high purity; e.g. Aldrich's Gold Standard grade)

Methanol (high purity; e.g., Aldrich or Burdick & Jackson's most commercially available grade)

Operating Procedures

This procedure is based on the Current Protocolsin Molecular Biology book.

1) Prepare acidified acetone:

Acetone 120 ml

HCl 10ul

Mix to make a 1 mmol/L solution.

2) Preparation of precipitant: Mix equal volumes of acidified acetone and methanol together. Freeze at -20℃.

3) Vacuum centrifuge and concentrate the protein solution to at least 0.1 ml.

4) Add 5 times the amount (by volume) of precipitant to the aqueous protein solution. Mix thoroughly and incubate overnight at -20°C in the refrigerator.

5) Centrifuge for 30 min at 4°C in a microcentrifuge at maximum speed.

6) Carefully remove the supernatant with a fine Pasteur pipette. Place the precipitate in a fume hood and blow with nitrogen for 1h until dry.

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