Figure. 4:(A) Analysis of Bocillin FL-binding activities in protein fractions of VISA strains. Penicillin-binding proteins (PBPs) were isolated from cytoplasmic membranes ("Mbr", left side) and CWDS fractions ("CWDS", right side). Bocillin FL was added to 100 μg protein in a final concentration of 20 μM and incubated for 30 min at 37°C. SDS-PAGE was performed in NuPage gels (Invitrogen) using SDS/MOPS buffer. Gel band detection and quantitation was done as described in the Method section. In lanes 1 and 1*: VP32; in the reaction for lane 1*, a 15-fold concentration of penicillin G was added to 20 μM Bocillin FL to demonstrate penicillin-specific protein modification; lanes 2: Mu50-32; lanes 3: Mu50; lanes 4: HIP5827. (B) The 75 kD protein from the VP32 sample in lane 1 ("Mbr") was separated in a 2-DE gel yielding a double spot train for PBP2; (C) Sequence coverage for PBP2 analyzed by LC-MS/MS; identified trypsin-digested peptides are underlined.