Here we introduce a protocol for Triton X-114 extraction which we used in our recently-published paper (Taguchi et al., 2013). It is a versatile method to concentrate or partially purify hydrophobic proteins. The presented protocol is based on the protocol published by Bordier (Bordier, 1981) but more simplified and down-scaled for more small-scale and simpler use (Taguchi et al., 2013).
Triton X-114 (TX114) is a non-ionic detergent which has a relatively low clouding point at 22 °C and separates into detergent (Det) and aqueous (Aq) phase at temperatures above the clouding point. During phase separation, hydrophobic solutes in the TX114 solution are sequestered to the Det phase, while hydrophilic solutes are sequestered to the Aq phase. Utilizing this phenomenon, TX114 extraction is a very versatile technique to efficiently concentrate hydrophobic proteins, especially glycosylphosphatidylinositol (GPI)-anchored proteins like the prion protein (PrP), because they have substantial amounts of highly hydrophobic moieties. Besides, phase separation using TX114 tolerates a variety of conditions, e.g. different pH or relatively low concentrations of guanidine hydrochloride. Since the hydrophobic proteins are sequestered to the Det phase as long as the phase separation occurs, and if the hydrophobicity of the protein of interest is not affected by pH or denaturant, this technique can be also utilized to change buffers or to remove denaturants. When using enzymes or proteases which maintain activities in detergent solutions, TX114 can also be used to separate hydrophobic from the water-soluble hydrophilic moieties upon enzymatic digestion of proteins, as done by us using in vitro digestion of PrP with phosphatidylinositol-specific phospholipase C (Taguchi et al., 2013).
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