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Shaking creates foam, introduces air bubbles into the solution, and can physically denature the peptide molecules

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The contents were vortexed and centrifuged at 8000 rpm for 5 minutes and the supernatant was analyzed for SOD activity by mixing with 50mM Tris-HCl buffer with 1.0 mM EDTA, pH 8.2 and 2.0 mM pyrogallal (in 50mM HCl)