A previously published fluorescence-based assay was utilized to monitor the enzymatic hydrolysis of 4-methylumbelliferyl heptanoate (4-MUH) (Sigma Aldrich).88 (link),92 (link) The recombinant Ag85A and Ag85C proteins were purified as previously described. Purified proteins were thoroughly dialyzed against 50 mM sodium phosphate (pH 7.5) buffer, and all assays were performed in triplicate on a Synergy H4 Plate Reader (Biotek) with λexcitation = 360 nm and λemission = 450 nm.
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Fluorescence-based Enzymatic Hydrolysis Assay
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Corresponding Organization : University of Nebraska Medical Center
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Variable analysis
independent variables
- Recombinant Ag85A and Ag85C proteins
- Enzymatic hydrolysis of 4-methylumbelliferyl heptanoate (4-MUH)
- 50 mM sodium phosphate (pH 7.5) buffer
- Assays performed in triplicate
- Synergy H4 Plate Reader (Biotek) with λ_excitation = 360 nm and λ_emission = 450 nm
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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