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Previous reports have indicated that the interaction of the functional -NO 2 group of TNBS with specific flavoproteins results in the formation of the nitro anion free radical, which in the presence of molecular oxygen yield the superoxide anion and hydrogen peroxide

After that, the mixture was transferred into the column and pre-washed with wash buffer (PBS, contained 0, 10, or 30 mmol/L imidazole, pH = 7.6), followed by elution buffer (PBS, contained 300 mmol/L imidazole, pH = 7.6)

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27.Silveira SQ, da Silva LM, de Campos Vieira Abib A, et al

The key consideration is preparation method