title: Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials.
abstract: Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.
authors.affiliation: Department of Advanced Technology, The MITRE Corporation; hfarris@mitre.org.
authors.forename: M Heath
authors.initials: MH
authors.lastname: Farris
authors.affiliation: Center for Biomedical Engineering, Brown University.
authors.forename: Kara A
authors.initials: KA
authors.lastname: Ford
authors.affiliation: The MITRE Corporation.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Doyle
mesh_terms: D000880:Anthraquinones / Q000737:chemistry*; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001681:Biological Assay; D010457:Peptidoglycan / Q000378:metabolism; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000880:Anthraquinones; D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D010457:Peptidoglycan; C024758:Remazol Brilliant Blue R