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Additional Information How to cite this article : Sabado, V

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The diagnostic approach: Run time course: Remove samples at 1h, 2h, 4h, overnight Analyze by SDS-PAGE Look for: Extra bands (degradation products), decreasing band intensity (target destruction) The solutions: Failure 3: Protease Inhibition (Your Buffer Kills the Enzyme) The problem: Proteases have specific requirements and sensitivities: Common mistakes: IMAC elution buffer: High imidazole inhibits thrombin Reducing agents: DTT reduces thrombin activity Metal chelators: EDTA kills metalloproteases Wrong pH: All proteases have pH optima The solution: Always buffer exchange into protease-compatible conditions before cleavage

It also inhibited inflammatory cytokines and decreased Desulfovibrio and Helicobacter , while increasing Lactococcus (Yang et al., 2017)

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