Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

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 complement...

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Bibliographic Details
Published in:Journal of visualized experiments 2016-04 (110), p.e53819-e53819
Main Authors: Farris, M Heath, Ford, Kara A, Doyle, Richard C
Format: Article
Language:English
Subjects:
Anthraquinones - chemistry
Anti-Infective Agents - pharmacology
Bacillus subtilis - drug effects
Bacillus subtilis - metabolism
Bacterial Proteins - metabolism
Biological Assay
Immunology
Peptidoglycan - metabolism
Reproducibility of Results
Salmonella enterica - drug effects
Salmonella enterica - metabolism
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Summary: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.
ISSN:1940-087X
1940-087X
DOI:10.3791/53819