RFC4GAT size exclusion chromatography. The molecular weight of RFC is 220 kDa, with each subunit as follows: Rfc1 65 kDa, Rfc2 40 kDa, Rfc3 38 kDa, Rfc4 36 kDa, and Rfc5 40 kDa. (A) An elution profile from Superdex 200 of RFC4GAT (1.6 mg) is shown. The complex was stored in 30 mM HEPES pH 7.5, 0.5 mM EDTA, 300 mM NaCl, 2 mM DTT, and 10% glycerol at −80 °C. Intensities (absorbance at 280 nm) are plotted as a function of elution volume and are an arbitrary scale. (B) SDS–PAGE (12%) analysis of fractions from the elution in panel A is shown. Lanes are labeled by elution volume (mL) and the input lane is the sample loaded onto the column. (C) An elution profile from Superdex 200 of RFC4GAT (2.2 mg) that was stored in 30 mM HEPES pH 7.5, 0.5 mM EDTA, 300 mM NaCl, 2 mM DTT, 10% glycerol, and 750 mM maltose at −80 °C is shown. Intensities (absorbance at 280 nm) are plotted as a function of elution volume and are an arbitrary scale. (D) SDS–PAGE (12%) analysis of fractions from the elution in panel C is shown. Lanes are labeled by elution volume (mL) and the input lane is the sample loaded onto the column.