Gst activity assay kit

The detection method of GST activity was modified from a GST activity assay kit from Cayman Chemical, Ann Arbor, MI, USA (item no. 703,302). After cell transfection for 24 h, cells were collected in GST store buffer (100 mM KH₂PO₄ (J.T.Baker, Radnor, PA, USA), 2 mM EDTA (Thermo Fisher Scientific)) and sonicated on ice. After centrifugation at 10,000× g for 10 min, 4 ℃, then the supernatant was further centrifuged at 100,000× g for 1 h, 4 ℃. The supernatant (cytosol fraction) was analyzed for GST activity in a 96-well plate. Twenty µL supernatant was added into a well with assay buffer (75 mM KH₂PO₄ (J.T.Baker), 0.075% Triton X-100 (J.T.Baker), 2 mM L-glutathione-reduced form (Alfa Aesar, Haverhill, MA, USA) on ice. After adding substrate 1-chloro-2,4-dinitrobenzene (CDNB) (Alfa Aesar) and mixing well, the OD340 nm values were read by a spectrophotometer at 25 ℃ for 20 min with 1 min interval. GST activity was calculated as [(OD340/min)/0.00503 µM⁻¹] × [0.2 mL/0.02 mL] = nmol/min/mL, then divided by protein concentration (mg/mL) to get the final GST activity (nmol/min/mg).

GST activity was measured using a GST activity assay kit (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. HepG2 cells were seeded with MEM in 96-well plates at a density of 1 x 10⁴ cells in each well and were treated with 10 μM B[a]P in the absence or presence of 40 μM silymarin for 48 h. The relative activation of GST was measured by using an Infinite 200 PRO multi-well plate reader (Tecan, Mannedorf, Switzerland).