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Cleavage Phase (During Processing) Buffer exchange first: Move from elution buffer to cleavage buffer Never assume elution conditions are compatible Start with pilot experiments: Small scale (100 g) Time course (1h, 2h, 4h, 16h) Multiple protease ratios (1:50, 1:100, 1:200) Monitor by SDS-PAGE: Look for: Band shift, new bands, loss of material Quantify cleavage efficiency Scale up conservatively: Optimal conditions at small scale may not translate directly Verify at each scale Post-Cleavage Phase (Cleanup) Remove uncleaved protein: Subtractive affinity (if cleaved protein doesn't bind) Ion exchange (if charge difference sufficient) Size exclusion (if size difference sufficient) Remove protease: Use orthogonally-tagged protease Or: Accept co-purification if protease doesn't interfere Verify final product: SDS-PAGE: Single band

Lury KM, Castillo M

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2016;113:E6806e12