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In brief, 50 g protein aliquots from each sample were mixed with denaturing buffer (8 M Urea, 0.1 M TrisHCl pH 8.5), then each aliquot was reduced and alkylated via sequential 20-min incubations with 5 mM TCEP (tris(2-carboxyethyl)phosphine) and 10 mM iodoacetamide at room temperature in the dark while being mixed at 1200 rpm in an Eppendorf thermomixer

Water is also known to have some filtration properties if it is present in the appropriate quantities, so there may be an additional benefit in this regard