Protease assay:
Proteolytic activity was determined, as described earlier [22Kaman WE, Hulst AG, van Alphen PT, et al. Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillusspecies. Anal Chem. 2011;83:25112517.[Crossref], [PubMed], [Web of Science ], [Google Scholar]], by incubating 16 M FRET-peptide with varying concentrations of PA83 or LF (both List Laboratories) in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) using blackwell clear bottom 96-well plates (Corning). …
Cleavage characteristics of PA83
20 g/mL PA83 was incubated with 16 M FRET-peptide PEK-054 (FITC-NleKKKKVLPIQLNAATDK-KDbc) [20Cummings RT, Salowe SP, Cunningham BR, et al. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease. …
LC MS/MS:
Prior to LC MS/MS analysis 10 g PA83 was digested with 0.5 g trypsin (Difco, BD Biosciences) for 3 hr at 37C. The trypsinized sample was analyzed by MS/MS using the data dependent LC-auto MS/MS mode. …
SDS-PAGE:
PA83 protein (2 g) was mixed 1:1 with 2x Laemmli sample buffer (Biorad), incubated for 5 min at 95C and loaded onto a 10% SDS-PAGE gel. After electrophoresis for 1 hr at 22 mA (max. 160 V) the gel was stained overnight with PageBlue staining solution (Fisher Scientific) and washed two times to visualize bands.
Specificity testing PA83
20 g/mL PA83 was incubated with 15 g/mL B. anthracis protective antigen antibody-[HRP] (-PA, Novusbio, BAP0102), 15 g/mL human IgG antibody-[HRP] (-human IgG, Sigma Aldrich, A0170) or PBS (solvent of the used antibodies) in HEPES buffer for 5 hr at room temperature. Next, 1 L PEK-054 (800 M) was added to the reactions and increase in fluorescence was measured at 37C for 60 min as described above.
Author did not indicate which specific lethal factor was utilized. List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).