Figure 2.

Comparison of Bocillin-FL labeling of PBPs using pretreatments with various permeabilization reagents. Live E. coli MG1655 were pretreated with 50 μL of PBS, Tris, Tris-EDTA (200 μM), or Tris-PMBN (200 μM) for 30 min, followed by incubation with 25 μM Bocillin-FL in either PBS (left lane) or Tris (remaining lanes) for 30 min. (A) An SDS-PAGE analysis showed the labeling of the entire PBP profile when cells were pretreated with Tris-EDTA or Tris-PMBN. The # indicates putative PBP assignments. One of three biological replicates is shown. (B) A comparison of the gel-band fluorescence showed significant increases in labeling for most PBPs when cells were pretreated with Tris-EDTA or Tris-PMBN. Mean fluorescence values that were corrected for protein loading differences via Coomassie stain from triplicate data sets for each PBP compared using one-way ANOVA multiple comparisons in GraphPad Prism [see the Methods section; P-values, 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)].