FIGURE 1.

SDS-PAGE (A) and Western blot (B) analysis of fractions obtained from purification of recombinant rat UPF0586 protein. Rat UPF0586 protein was purified to homogeneity by affinity chromatography on nickel-Sepharose (HisTrap HP) as described under “Experimental Procedures.” For the SDS-PAGE analysis, 7.5 μl of sample from each fraction was loaded onto a 10% gel and electrophoresed, and the resulting gel was then stained with silver (11). For the Western blot analysis, 7.5 μl of each fraction was loaded onto a 10% gel, electrophoresed, and blotted to nitrocellulose membrane, which was then sequentially probed with a mouse primary antibody against His₆ tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. M, prestained protein marker; L, cell-free lysate of COS-7 cells overexpressing the recombinant enzyme; AL, 4-fold diluted lysate applied on the column; FT, flow-through; W, wash. Fractions 30–300 were eluted with the indicated concentrations of imidazole.