Glutathione s transferase assay kit

Total glutathione-S-transferase (GST) activity (both cytosolic and microsomal) was measured in 10 mg of liver tissues using the Glutathione-S-transferase Assay kit (703302, Cayman Chemicals, MI, USA) according to the kit protocol. Data from the activity assay was normalized to protein concentration estimated using the Lowry protein quantification assay.

All samples were diluted 1:5 in HEPES buffer. The total GST activity was determined using the Glutathione S-Transferase Assay Kit (Cayman Chemical, item no. 703302) according to the manufacturer’s instructions. The absorbance was recorded every minute at 340 nm for 60 min and the linear range from minute 1 to 9 was used for calculation of the GST activities (nmol/min/ml). Measurement of the GST activity was repeated using a different GST assay kit (Abcam, ab65325) and newly sampled hooded seal tissues (March 2021). The tissue was homogenized in Assay buffer and the concentration of all samples was adjusted to 3.9 mg/ml. The assay was performed according to the manufacturer’s instruction using a 1:5 dilution of the samples.

Plasma GST activity was determined using the standardized Glutathione S-transferase Assay Kit (Cayman Chemical Co. Item No. 703302). The analyses were performed on 96-well plates according to the methodology provided by the manufacturer. Three non-enzymatic background samples were prepared by placing 170 μl of Assay Buffer and 20 μl of glutathione in the wells. Then, three samples of the positive control containing equine liver GST were prepared by adding 150 μl of Assay Buffer, 20 μl of glutathione, and 20 μl of control GST. The remaining wells were filled with 20 μl of test plasma, 150 μl of Assay Buffer, and 20 μl of glutathione. The reaction was started by adding 10 μl of 1-chloro-2,4-dinitrobenzene (CDNB) to all wells in the plates. The plates were carefully shaken for a few seconds on a shaker. Five absorbance measurements were made every minute at 340 nm using a plate reader (Multiskan RC version 6.0, Labsystems). GST activity was calculated by analyzing the change in absorbance per minute corrected for non-enzymatic background. The calculated GST activity was expressed in nmol·min⁻¹·ml⁻¹.

SOD activity was assessed using an assay that utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine using a superoxide dismutase assay kit colorimetrically at 450 nm (Cayman, Ann Arbor, MI, USA). One unit of SOD was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.
Total GST activity was measured by quantifying the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione using a glutathione S-transferase assay kit colorimetrically at 340 nm (Cayman).

Glutathione S-Transferase assay kit was purchased from Cayman chemicals. For analyzing GST activity in QC treated and untreated cell lysates of A549 and NCI H520 cells, 100 μg of cell lysate were taken and diluted in sample dilution buffer to make sample volume of 20 μl as recommended in manufacturer’s instruction manual for performing the assay. For confirmation of the effect of Quinacrine and its comparison to the effect of well-known inhibitor Ethacrynic acid, three different concentrations (500 nM, 1 μM and 2.5 μM) of both compounds were added into separate wells along with reduced glutathione and GST control protein provided with kit and incubated at 25°C for 10 mins prior to adding CDNB. This assay was performed separately. Both the assays were performed in triplicates and as per manufacturer’s instruction and the GST activities of samples were calculated accordingly.

The reaction contains 1000 μL of the solution, in which 75 μL of Assay buffer, 10 μL of the homogenized sample, 10 μL of glutathione were added. To initiate the reactions, 5 μL CDNB was added to each well in triplicates to microplate at room temperature. To measure the enzyme kinetics, a 96 well microplate was loaded with reaction solution and shaken for 10 s. After 60 s of lag time, the absorbance was read at 340 nm and the homogenates were read for 20 min at 37 °C with a microplate reader. The GST activity was determined from the extinction coefficient of 0.0096 µM⁻¹ cm⁻¹ for CDNB. The enzyme activities were expressed as micromolar per milligram protein per minute (µmol/min/mg protein). For the determination of GST and AChE enzyme, the Glutathione-S-Transferase Assay Kit was procured from Cayman Chemical, 1180 E, Ellsworth Road, Ann Arbor, MI, USA.