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SDS-PAGE and Western Blot analysis Twenty L of sample containing 5 mg of purified rhGPx3 mutant was mixed with 20 L of loading buffer (20 mM Tris-HCl, 8% SDS, 10% -mercaptoethanol, 30% glycerol and 0.004% bromophenol blue at pH 6.8) and heated in boiling water for 5 min