Small-scale Triton X-114 Extraction of Hydrophobic Proteins
Bibliographic record
Abstract
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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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.002 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.003 |
| Insufficient payload (model declined to judge) | 0.012 | 0.016 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".