His-FLAG Mcm10. Mcm10 with a hexahistidine tag at the N-terminus and a 3X FLAG tag at the C-terminus, was purified by first applying clarified extract from 72L E. coli overexpression cells to a column containing 10 ml Chelating Sepharose Fast Flow (GE Healthcare) charged with 50 mM NiSO4. The column had been previously washed with binding buffer (20 mM Tris-Cl pH 7.9, 5 mM imidazole, 500 mM NaCl, 0.01% NP-40) and the E. coli extract was adjusted to approximate the ionic strength of the binding buffer and applied to the column at 1 ml/min. The column was washed with binding buffer and then eluted with the same buffer containing 375 mM imidazole. The eluted material was then applied to a column containing 6 ml ANTI-FLAG M2 Affinity Gel (Sigma) at 0.1 ml/min. The FLAG column had been previously equilibrated in FLAG buffer (20 mM Tris-Cl pH 7.5, 10% glycerol, 500 mM NaCl, 1 mM DTT, 1 mM MgCl2, 0.01% NP-40) and after loading, the column was washed with 50 ml of FLAG buffer. The bound material was eluted with 20 ml FLAG buffer containing 0.2 mg/ml 3X FLAG peptide (EZ Biolab, Carmel, Indiana USA) and collecting 1.5 ml fractions. The elution buffer was applied to the column in two 6 ml increments pausing 30’ after each increment followed by 3 ml increments with 30’ pauses until the elution was complete. Eluted material was aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.