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158 Godat et al.<br />

3. Sonicate samples on ice. Sonicate with 5s pulse plus 5-s gap until cells are<br />

completely lysed.<br />

4. For large-scale column purification, clear the lysate before loading the column by<br />

centrifuging at 10,000 × g for 30 min at 4ºC and discard pellet.<br />

3.4.2. Column Preparation<br />

1. Determine the column volume required to purify the protein of interest. In most<br />

cases, 1 ml of settled resin is sufficient to purify the amount of protein typically<br />

found in up to 1Lofculture (cell density of OD 600 nm

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