A modified CAS liquid assay for the determination of Cd-chelating ability was improved from a Fe-CAS assay [66 (link)], which is conducted as follows: 4 mL 50 mM CAS, 2 mL 10 mM dipy, 1.5 mL 1 × 10−3 M HDPB, and 100 mM NaOH was titrated till the system turned green. After being placed stably for 5 min, a 5 mL buffer solution of sodium borate sodium hydroxide (pH 11.0) was added. Then diluted with H2O to a constant volume of 25 mL. The change of optical density value (OD) was measured at 602 nm within 40 min using a Varioskan Flash Multiplate Reader (Thermo Fisher, MA, USA). For the CAS plate assay, 2 g/100 mL agar was added to the solution system mentioned above. The chelation rate was calculated as (H2O-treated OD value–treatment OD)/(H2O-treated OD value).
Twenty AAs (1 mM) were respectively added to the CAS reaction system to determine the OD value. HLA-1-1 was cultured for 7 days with/without Thr to obtain the supernatant after centrifuging at 10,000 r/min for 10 min, then stored at −50 °C targeting concentrate supernatant via vacuum freeze-drying. Strains were harvested from fragments of HLA-1-1 obtained from the culture media after centrifuging at 10 000 r/min for 10 min, then put in the bead beater for milling a couple of times after adding magnetic beads.
Twenty AAs (1 mM) were respectively added to the CAS reaction system to determine the OD value. HLA-1-1 was cultured for 7 days with/without Thr to obtain the supernatant after centrifuging at 10,000 r/min for 10 min, then stored at −50 °C targeting concentrate supernatant via vacuum freeze-drying. Strains were harvested from fragments of HLA-1-1 obtained from the culture media after centrifuging at 10 000 r/min for 10 min, then put in the bead beater for milling a couple of times after adding magnetic beads.
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