Purification and MS analysis of <i>N</i>. <i>meningitidis</i> EF-P(<i>Nm)</i>.

<p>Proteins in each purification step were analyzed by 10–20% SDS-PAGE and stained with SimplyBlue SafeStain. (A) Lane 1, molecular mass standards; lane 2, <i>N</i>. <i>meningitidis</i> crude cell extract; lane 3, after DEAE-Sephacel column; lane 4, after HiTrap Q HP column; lane 5, endogenous EF-P(<i>Nm</i>) purified on a HiTrap Butyl HP column; lane 6, MagicMark molecular mass standards (Life Technologies). (B) The polyclonal antibody against EF-P(<i>Ec</i>) cross-reacts with EF-P(<i>Nm</i>). The EF-P proteins in the <i>N</i>. <i>meningitidis</i> cell extracts and the column chromatography fractions were monitored by western blotting with the antibody. Lane 1, <i>N</i>. <i>meningitidis</i> crude cell extract; lane 2, MagicMark molecular mass standards. The arrow designates EF-P(<i>Nm</i>). (C) MALDI-TOF MS and SDS-PAGE of the endogenous EF-P(<i>Nm</i>), shown in the left and right panels, respectively. (D) MALDI-TOF MS and SDS-PAGE of the recombinant EF-P(<i>Nm</i>), shown in the left and right panels, respectively.