The standard assay mixture consisted of 0.6 mM 2,2′-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) (ABTS) (Sigma-Aldrich, Tokyo, Japan), 3 units of peroxidase from horseradish, 1 mM of 4-amino-4-deoxyarabinitol, 47.1 mM of potassium phosphate, pH 7.0, and an appropriate amount of sample containing NecC in a final volume of 1 mL.
Cytotoxic activity was evaluated by the brine shrimp (Artemia salina) test in triplicate with appropriate amounts of samples dissolved in DMSO (1% final volume) to reach final concentrations of 100, 10 and 1 μg/ml, using 10 freshly hatched larvae suspended in 5 ml of artificial seawater (Meyer [1982]).
A total of 100 μl of stool specimen was diluted in appropriate amounts of sample buffer included in the commercial kit (DakoCytomation Ltd., Cambridgeshire, UK).
The remaining ER proteins were mixed with an appropriate amount of 5×SDS sample loading buffer, boiled for 4 min, and spun at 13000× rpm for 5 min. The supernatant was stored at −20C for SDS-PAGE.
An appropriate amount of the prepared samples was reflux with 80%% ethanol at a solid liquid ratio of 1 10 (m/v) at 80 °C for 2 h.
An appropriate amount of the ground sample was pelletized using a SPECAC bolt and nut pelletizer to give a transparent pellet.
This level of performance allows for predictable sequence coverage beyond what is reported in Table 1 with the appropriate amount of sequencing per sample (see additional coverage analysis in Additional file 3: Table S4).
An appropriate amount of freeze-dried sample was dispersed in phosphate buffer solution (PBS) at the pH of 7.4 and placed in the dialysis bag (cut-off 6000 8000 Da).
The purification procedures of the samples were carried out by adding appropriate amount of ethanol into the samples and centrifuging at 4000 rpm for 10 min. After that, the precipitates were collected and washed twice with chloroform to remove precursor and surfactant residuals.
To prepare samples, an appropriate amount of 4× loading buffer was added, and sample was boiled for 10 min, cooled down on ice, and centrifuged.
