Urea assay method

1. Preparation of samples for urea concentration assay

1. A single colony of cells transformed with mock, pTrc-rocF or pTrc-rocF-Arg box was inoculated into 3mL of LB with kanamycin and grown to saturation at 37℃.
2. The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).
3. The culture was induced with 1mM IPTG at 37℃ for 1 hour.
4. 2mL of culture was centrifuged at 9,000 rmp for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.

2.Urea concentration assay

Urea concentrations of the samples were determined colorimetrically withDIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems.
Each sample was assayed in triplicate.

1. 10 µL of the supernatant fluid from each sample, 10 µL blank(LB),and 10 µL standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.
2. 200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
3. Optical density at 450nm was read and urea concentration (mg/dL) of the sample was calculated as
   .
   ODSAMPLE, ODBLANK and ODSTANDARD are OD450 values of sample, standard and blank, respectively.

Standard curve for coloring reaction in urea assay

To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.

Fig.1 Standard curve for coloring reaction in urea assay