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For cleavage of the N-terminal hexahistidine-tag, TEV protease was added to the pooled elution fractions and dialyzed overnight into dialysis buffer at 4 C (25 mM Tris pH 8.0, 100 mM NaCl, 4 mM MgCl 2 , 10 M GDP, 1 mM TCEP, and 5 % glycerol)

The bigger factor is sticking to the same time daily, not which half of the day you choose

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