Practical Tips for Sample Preparation

One inherent property of peptides that interact with membranes is that self-association or even aggregation will interfere with solubilization by organic solvents or micelles. The preparation, purification and sample preparation of extremely hydrophobic (often transmembrane) peptides is nontrivial and has been addressed by only a few papers [74—79]. 

The first problem encountered once the peptide has been successfully synthesized is that standard purification protocols fail. Although very hydrophobic peptides are soluble in acids such as TFA, these harsh conditions are not suitable for purification, because they can reduce column life times and denature native protein structures. Hence residual acid has to be removed, and many peptides can then be redissolved in mixtures of water and tert-butanol. Peptides with a strong tendency to aggregate may be dissolved either in trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), mixtures of 1-propanol and 1-butanol, 20% acetic acid or 70-90% formic acid. 

A general procedure has been reported by Killian et al. for the incorporation of hydro-phobic peptides into micelles [75]. Therein, the peptides were first dissolved in TFA for deaggregation, dried under nitrogen and redissolved as 5 mM (clear) solutions in TFE or HFIP. The solution is subsequently diluted 1 1 by addition of an aqueous solution containing a varying SDS concentration (typically 500 mM, depending on the protein concentration). Water is then added to yield a 16 1 ratio of water to TFE (HFIP) by volume. Upon addition of excess water the peptide loses its solubility, but at the same time the 

Although detailed protocols for preparation of small unilamellar vesicles (SUVs) are published [80-82], their preparation is more time-demanding and complicated. In contrast, bicelles are prepared similarly to micelles with little effort. 

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