Detection of UmuC location by immuno-electron microscopy and solubility in cell lysates.

<p>(A–B) Immuno-electron microscopy of plasmid-expressed UmuC in the presence of <i>umuD</i>(C24D G25D) and <i>umuD</i>' in the <i>E</i>.<i>coli</i> strain RW1394. Sectioned electron-microscopy grids were incubated with anti-UmuC antibody and subsequently labeled with 30 nm gold beads conjugated to a secondary goat-anti-rabbit antibody. (A) Representative micrographs. Although the labeling density was low, gold beads were clearly visible for cells expressing UmuC, whereas control cells lacking UmuC showed virtually no beads. (B) Quantification of the proportion of beads associated with the membrane each sample. Beads found to be within 100 nm of the cell membrane were classed as membrane-associated. (C) Levels of soluble UmuC detected by western blotting (<i>E</i>. <i>coli</i> strain, TCH03). The two images are identical, except that upper panel is a darker exposure of the lower panel. UmuC was expressed alone, or co-expressed with UmuD' or UmuD(K97A). The images clearly show that UmuC is most soluble when co-expressed with UmuD' and least soluble when co-expressed with UmuD(K97A).