GlucosinolatesAnalysis.md

Glucosinolates

GSLs were extracted from On-Hold mg of homogenized plant material using 500 ml of hot 70% (v/v) methanol, and 10 μl of sinigrin was added as internal standard. The extract was vortexed and incubated at 70 °C for 45 min, vortexing twice during the incubation. The samples were left to cool and centrifuged at maximum speed for 5 min at room temperature. The supernatant was transferred to prepared columns containing 0.5 ml of DEAE Sephadex A-25, washed twice with 0.5 ml of sterile H2O, and subsequently twice again with 0.5 ml of 0.02 M sodium acetate buffer. With a new tube placed underneath each column, a layer of 75 μl of sulfatase was placed onto the column. The samples were left at room temperature overnight, and the resulting desulfo-GSLs were eluted twice with 0.5 ml of sterile H2O, followed by a final elution by 0.25 ml. The eluates were combined, vortexed, centrifuged for 5 min, and 200 μl of the supernatant were transferred to HPLC vials. The desulfo-GSLs were resolved by HPLC (Spherisorb ODS2, 250 3 4.6 mm, 5 μm; Waters) using a gradient of acetonitrile in water and detected by UV absorption at 229 nm. The GSLs were quantified using the internal standard and response factors as described in Dietzen et al. (2020).