5430r centrifuge

Total superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) activities were determined in crude extracts of leaf tissues. Leaf samples (200 mg) were ground in liquid nitrogen with insoluble polyvinyl pyrrolidone, extracted in 0.066 M potassium-phosphate buffer (pH 7.4) containing 0.5 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride in dimethyl sulfoxide, and then centrifuged for 20 min at 8000 rpm and 4 °C using a 5430R centrifuge (Eppendorf, Germany). The total SOD activity was determined according to Beauchamp and Fridovich [71 (link)]. The reaction medium (2 mL) contained 10 μL of supernatant, 1.75 mL of 50 mM Tris-HCl buffer (pH 7.8), 0.2 mL of 0.1 M DL-methionine, 0.063 mL of 1.7 mM Nitro Blue tetrazolium (Fermentas, Waltham, MA, USA), 0.047 mL of 1% Triton X-100 and 0.060 mL of 0.004% riboflavin. The reaction proceeded under LED lamps (I = 232 μmol photons/m⁻²s⁻¹) for 30 min. The absorption was measured at 560 nm using a Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, USA). Peroxidase activity was determined as previously described [72 (link)]. The reaction mixture contained 50 μL of supernatant, 1.95 mL of 0.066 M potassium-phosphate buffer (pH 7.4), 200 μL of 7 mM guaiacol and 250 μL of 0.01 M H₂O₂. The absorption was measured at 470 nm using a Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Studies were performed in two different laboratories with slightly different instrumentation as follows: Sample tissue was homogenized using (1) equal parts sample and DI water using a Waring (Lancaster, PA, USA) model LB10S variable speed steel bowl lab blender, or (2) homogenized frozen with a Robot Coupe (Jackson, MS, USA) RSI 2Y1 laboratory grade blender by incorporating a small amount of dry ice. Samples were agitated either manually or with an ATR (Laurel, MD, USA) RKVSD Rotamix rotating inverter. All extracts were centrifuged with either a Thermo Fisher Scientific (Waltham, MA, USA) Sorvall Evolution RC centrifuge or Eppendorf (Hamburg, Germany) 5430R centrifuge. SBSE was performed at room temperature with GERSTEL Twister stir bars in 10 mL headspace vials on a 20 position magnetic stir plate. Stir bars were thermally desorbed using a TDU thermal desorption unit with a CIS 4 programmed temperature vaporizing inlet and analysis automated using an MPS 2 autosampler with Maestro software (GERSTEL, Linthicum, MD, USA). Lastly, an Agilent (Santa Clara, CA, USA) 7890 GC interfaced with either an Agilent 5975 or 5973 MS was used.

SDS-PAGE was analyzed using the method of Li, et al. [18 (link)] with some modifications. Before digestion, crayfish meat gels (4 g) were mixed sodium dodecyl sulfate (SDS) solution (1%, w/v, 36 mL) and homogenized (5000 rpm, 3 min) with a T18 homogenizer (IKA, Schwarzwald, Germany). The mixture was centrifuged (5000 rpm, 5 min, 6 °C) by a 5430R centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was separated and adjusted to a uniform protein concentration level. After digestion, 0.4 mL of the supernatant from the digested sample was used for analysis. Electrophoresis was performed by a DYCZ-24KF electrophoresis apparatus (Liuyi Biological Technology Co., Ltd., Beijing, China) at 170 V for about 3 h.