Protocol Trichloroacetic Acid (TCA)Precipitation of Proteins Andrew J.Link and Joshua LaBaer INTRODUCTION Cell or tissue extracts of proteins often contain interfering substances that must be removed.A number of methods are available for concentrating dilute protein solutions and simultaneously removing contaminating substances.Trichloroacetic acid (TCA)/acetone precipitation,as described here,is often used for this purpose.MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution ’s Environmental Health and Safety Of fice for proper handling of equipment and hazardous materials used in this protocol.Reagents Acetone Protein sample Trichloroacetic acid (TCA)(Sigma-Aldrich T9159)Prepare a 100%(w/v)solution by dissolving 2.2g of TCA in 1mL of H 2O.Then prepare a 10%solution by adding 0.1mL of 100%TCA solution to 0.9mL of H 2O.Keep both solutions ice cold.Equipment Microcentrifuge Vacuum evaporator (e.g.,SpeedVac)METHOD    1.Add 0.11volumes of ice-cold 100%TCA to the protein sample.
2.Place on ice for 10min.
3.Add 500µL of ice-cold 10%TCA to the sample.
4.Place on ice for 20min.
5.Centrifuge at 20,000g for 30min.
6.Carefully remove the supernatant.
Aspiration of the supernatant with a pipette or a disposable transfer pipette works well.Avoid disturbing the protein
pellet.
7.Add 500µL of acetone.Very gently rock the tube once or twice to rinse the tube and pellet.
Adapted from Proteomics:A Cold Spring Harbor Laboratory Course
Manual ,by Andrew J.Link and Joshua LaBaer.CSHL Press,Cold
Spring Harbor,NY,USA,2009.
Cite as:Cold Spring Harb Protoc;2011;doi:10.1101/pdb. ©2011Cold Spring Harbor Laboratory Press 993P R O T O
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8.Centrifuge at 20,000g for 10min.Very carefully remove the supernatant to avoid disturbing the
protein pellet.
Aspiration of the supernatant with a pipette or a disposable transfer pipette works well.
9.Dry the protein pellet in a vacuum evaporator (typically <5min).
The pellet is now ready for resuspension in a mass spectrometry compatible buffer.
994Cold Spring Harbor Protocols P R O T O C O L S w w w .c s h p r o t o c o l s .o r g
doi: 10.1101/pdb.prot5651
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