β-Lactam probes covalently bind to Mtb proteins, enabling fluorescent detection. Lysates were treated with a probe, resolved by SDS-PAGE, and imaged. Three autofluorescent bands (X) were observed with Bocillin-FL but not with Cy5 probes. Labeling was reduced by pretreatment with the parent antibiotic (left lanes). Pen, penicillin G; Azt, aztreonam; CX, cephalexin; Mero, meropenem. Red star: Targets bound and detected by Azt-Cy5, CephX-Cy5, or Mero-Cy5 (but not Bocillin-FL). BlaC migrates at 30 kDa and is denoted by gold triangles.