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Solutions for improving cleavage: More protease: But watch for non-specific cleavage Longer incubation: But protein may aggregate or degrade Higher temperature: TEV is maximally active at 34C, but your protein may not survive Fresh protease: Old protease loses activity Solutions for separating cleaved/uncleaved: Failure 5: Removing the Protease (Now You Have a New Contaminant) The problem: After cleavage, your sample contains: Cleaved target protein Uncleaved target protein Free tag Protease If the protease has the same tag as your protein (common with His-tagged TEV), standard subtractive purification won't workboth bind or both flow through together

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