Electrophoretic mobility and size-exclusion chromatography profile of RYMV and LMV VPgs. A, C. 15% SDS-PAGE of recombinant His-tagged RYMV and LMV VPgs recovered from the supernatant (SN) and from the cell pellet (CP) after E. coli cell extraction, and after imidazole gradient elution fractions (E1 to E5) obtained after loading a 1 ml affinity nickel column (GE Healthcare) with the soluble fraction of the bacterial lysate. Low molecular weight (LMW) protein standards for SDS PAGE (GE Healthcare) are shown. The expected molecular masses of 10.53 and 26.25 kDa respectively were indicated by broken lines. The proteins in the major band (indicated by an arrow) migrate with an apparent molecular mass of about 15 and 27 kDa, respectively. B, D. Elution profile of purified His-tagged VPgs from a Superdex 75 HR10/30 column (GE Healthcare) in 50 mM Tris-HCl pH 8, 300 mM NaCl, at a flow rate of 0.5 ml/min. The proteins were eluted in a major peak with an apparent molecular mass of about 17 and 40 kDa respectively as deduced from column calibration with low molecular weight protein standards for gel filtration (GE Healthcare).