Superoxide dismutase assay kit 706002

To determine the parameters of oxidative stress, hemolysates were prepared from whole blood taken from each rat into EDTA tubes. After the whole blood was centrifuged at 4000 rpm for 5 min, the plasma was separated, the remaining erythrocyte package was placed in a 0.5 mL clean glass tube and 2 mL isotonic NaCl was added and mixed. The supernatant fraction was discarded by centrifugation at 4000 rpm for 5 min. The last two steps were repeated 3 times. After the last repetition, 2 mL cold distilled water was put into the tube and erythrocytes exploded. The obtained hemolysates were stored at −80 °C until analysis.
In the erythrocytes obtained; superoxide dismutase (SOD) activity (Cayman Superoxide Dismutase Assay Kit 706002), catalase activity (Cayman Catalase Assay Kit 707002), glutathione peroxidase activity (Cayman Glutathione Peroxidase Assay Kit 703102), and malondialdehyde concentration (Cayman TBARS Assay Kit 10009055) determination were performed.

Samples taken were weighed by precision scales in accordance with SOD kit (Cayman Chemical Company Superoxide Dismutase Assay Kit 706002, E. Ellsworth Rd-Ann Arbor, MI, USA) procedure and their homogenates were prepared. Tris-HCI buffer (pH=7.4) was added to tissue samples to make a dilution at a rate of 1/10 (m/v). The samples were homogenized in a ball homogenizer (Retsch MM400) at 30 Hz frequency for 10 min by being kept in their coldness. The tubes taken from the homogenizer were centrifuged at 4000 rpm for 10 min. SOD activity was measured spectrophotometrically at 440-460 nm on the supernatants obtained as defined in the kit prospectus.

In order to evaluate if the iron treatment promotes oxidative stress in Chromobacterium violaceum, antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities were measured on total protein extracts. Catalase Assay Kit 707002 and Superoxide Dismutase Assay Kit 706002 (Cayman Chemical, Ann Arbor, MI) were used according to manufacturer’s recommendations to quantify the catalase and superoxide dismutase activity level, respectively. The total antioxidant activity was evaluated using the Antioxidant Assay kit from Sigma-Aldrich (CS0790) according to manufacturer’s instructions. The protein concentration values were used to normalize the enzyme activity. The experiment was performed in biological triplicate.