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The peptide must be fully dissolved in the organic solvent before any water is added, because adding water first produces a mixture in which the peptide dissolves more slowly and more organic solvent ends up being used than was necessary

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Cleaved protein was repurified from the cleaved fusion tag and TEV protease by loading on a Ni-NTA resin column and 5 ml of the flow through was applied to a size-exclusion column (HiLoad Sepharose 16/600 75 pg, GE Healthcare) on an ÄKTA Pure system (GE Healthcare) at RT