TCA precipitation principles
TCA precipitation principles

TCA (trichloroacetic acid) precipitation is considered the most efficient protocol for precipitating proteins from dilute solution. Surprisingly little is known of the mechanism. However Sivaraman, et al J. Protein Chem 16, 291-7 (1997) show that precipitation vs TCA concentration is a U shaped curve with optimum ppt at roughly 15% TCA and suggest that the mechanism is hydrophobic aggregation. Xu et al , J. Protein Chem 22, 669-75 (2003) support the hydrophobicity precipitation mechanism.

Bensadoun and Weinstein (Anal Biochem 70, 241-50 (1976) - heavily cited) show that an added carrier, deoxycholate (DOC), improves recovery. DOC is a detergent at neutral pH. Acidification titrates acidic groups dramatically reducing solubility. Chevallet, et al Proteomics 7, 1757-70 (2007) adapted this technique to proteomics by substituting sarkosyl for DOC and washing away precipitated sarkosyl with tetrahydrofuran (THF).

Note that sarkosyl hydrolyzes slowly in aqueous solution. Solutions should be less that ~2wks old (I haven't tested this rigorously).

My protocol includes a lipid soluble dye (Sudan Black) to mark the precipitate. THF dissolves the dye. If you're TCA precipitating several samples, gray color in the TCA wash is a useful indicator of "pellet recovery".

I've also precipitated fluorescent (dansylated) proteins. The fluorescence allows one to detect small pellets. Ideally one could dansylate a small protein (i.e. insulin) to serve as an internal recovery standard.

Note that THF is similar to ether, but has advantages of water miscibility and lower volatility.