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After protein quantification, loading buffer containing 10% SDS, 0.5% bromophenol blue in Tris-HCL (pH 6.8), and 10% -mercaptoethanol (M3148, Sigma) was added to each cell lysate and boiled at 95-100 C for 10 min

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Here's the key: DO NOT inject the water directly onto the lyophilized powder