The antioxidant capacity was determined by the reduction of radical monocation, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), according to the procedure described by Gião et al. [48 (link)]. The radical was obtained after the addition of 7 mmol/L of ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Saint Louis, MO, USA) to 2.45 mmol/L of a potassium persulfate solution (1:1 (v/v)). The mixture was left to react in the dark for 16 h. To obtain an absorbance of 0.700 ± 0.020 at 734 nm, the ABTS•+ solution was diluted using ultrapure water. For the reactions, 30 μL of each filtered and diluted extract was mixed with 3000 μL of the ABTS•+ solution. After 6 min, the absorbance was measured at 734 nm with a spectrophotometer (Metash, Shanghai, China) using ultrapure water as a blank. The ABTS•+ antiradical activity was calculated using Trolox solutions (Sigma-Aldrich, Buchs, Switzerland) with different concentrations ranging from 240 to 2000 μmol (linear regression: y = 0.0003x + 0.0094; R2 = 0.9989). The results are expressed as μmol of Trolox equivalents per gram (μmol TE/g).
Trolox solution
Manufactured by Merck Group
Sourced in Switzerland, United States
Trolox solution is a water-soluble vitamin E analog commonly used as an antioxidant in biochemical and cell culture applications. It functions as a free radical scavenger to protect samples from oxidative damage.
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Lab products found in correlation
Abts radical, by Merck Group (1 mentions)
Evolution 260 bio spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Catalase, by Merck Group (1 mentions)
Plan apo, by Nikon (1 mentions)
D glucose, by Merck Group (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Uv 2600i spectrophotometer, by Shimadzu (1 mentions)
Hlp 20uv, by Hydrolab (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
7 protocols using trolox solution
1
ABTS Antioxidant Capacity Assay
2
Antioxidant Capacity Determination by ABTS Assay
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The antioxidant capacity was determined by the reduction method of the ABTS•+ radical (Sigma-Aldrich, Saint Louis, MO, USA) according to Gião et al. [41 (link)]. For the reactions, 30 μL of each filtered and duly diluted extract were mixed with 3000 μL ABTS•+ radical. After 6 min, the absorbance was measured at 734 nm with a spectrophotometer in spectrophotometric units (Metash, Shanghai, China) using ultra-pure water as a blank. The ABTS•+ antiradical activity was calculated using Trolox solutions (Sigma-Aldrich, Buchs, Switzerland) with different concentrations in a range of 500–2000 μmol. Results were expressed as µmol Trolox equivalents per gram (μmol TE/g).
3
DPPH Radical Scavenging Assay
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The 2,2′-diphenyl-β-picrylhydrazyl radical (DPPH•) (Sigma-Aldrich, Steinheim, Germany) scavenging activity of the extracts was determined according to the method described by Hidalgo et al. [49 (link)]. For the reactions, 100 μL of each diluted extract was added to 2900 μL of a DPPH• solution (6 × 10−5 M in methanol and diluted to an absorbance of 0.700 at 517 nm). The resulting solutions were allowed to stand for 30 min in the dark at room temperature. Then, the absorbance was measured at 517 nm with a spectrophotometer (Metash, Shanghai, China) using methanol as a blank. The DPPH• scavenging activity was calculated using Trolox solutions (Sigma-Aldrich, Buchs, Switzerland) with different concentrations ranging from 80 to 680 μmol (linear regression: y = 0.0008x + 0.017; R2 = 0.9962). The results are expressed as μmol TE/g.
4
DPPH Radical Scavenging Activity Assay
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The radical scavenging activity of the extracts and fractions against the DPPH free radical was determined according to Brand-Williams [42 (link)], with slight modifications as described by Lis et al [17 (link)]. Briefly, 1900 µL of DPPH methanol solution (100 µM) was mixed with 100 µL of the sample (four different concentrations in the range between 10–500 µg/mL) or Trolox solution (Sigma-Aldrich; five different concentrations in the range between 10–200 µg/mL) in a cuvette. After 30 min, the absorbance was measured against methanol (blank) at 517 nm using the Evolution 260 Bio spectrophotometer (Thermofisher Scientific). The percentage of absorbance inhibition was calculated from the equation:
Inhibition (%) = 100 × [(Ablank − Asample)/Ablank], where Ablank and Asample are the absorbance values of the blank and test samples at t = 30 min, respectively.
To calculate the Trolox Equivalent (TE) of samples on DPPH, the slope of the sample linear curve, i.e., the absorbance inhibition (%) vs. concentration (µg/mL), was divided by the slope of the standard linear curve.
The IC50 value of extracts and fractions, defined as the concentration of sample necessary to cause 50% inhibition was determined from the sample linear curves as absorbance inhibition (%) vs. concentration (µg/mL), with Trolox used as a positive control.
Inhibition (%) = 100 × [(Ablank − Asample)/Ablank], where Ablank and Asample are the absorbance values of the blank and test samples at t = 30 min, respectively.
To calculate the Trolox Equivalent (TE) of samples on DPPH, the slope of the sample linear curve, i.e., the absorbance inhibition (%) vs. concentration (µg/mL), was divided by the slope of the standard linear curve.
The IC50 value of extracts and fractions, defined as the concentration of sample necessary to cause 50% inhibition was determined from the sample linear curves as absorbance inhibition (%) vs. concentration (µg/mL), with Trolox used as a positive control.
5
Single-Molecule TIRF Microscopy of gp32
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All of our single-molecule experiments were performed using a prism-based TIRF microscope that was equipped with a 532 nm laser and 100x NA, 1.4 oil-immersion objective (Plan Apo, Nikon), as previously described (6 (link),22 (link)). Experiments were carried out at room temperature (22°C) in a temperature-controlled laboratory that is stabilized to within ±0.5°F. Sample solutions were prepared using the standard imaging buffer unless otherwise specified. These solutions contained the oxygen scavenging and triplet quenching system used only for smFRET measurements: 165 U/ml glucose oxidase (Sigma), 0.8% (w/v) D-glucose (Sigma), 2170 U/ml Catalase (Sigma) in a Trolox solution (≥1 mM, Sigma). Protein (gp32) solutions at various concentrations containing these oxygen scavenging and triplet quenching system were incubated in a microfluidic sample chamber for 2–3 min before data acquisition. Unless otherwise specified, data were acquired without flushing the unbound proteins from the sample chamber. Data sets were generally collected on the same day for a given experiment (e.g. salt concentration dependence of a particular substrate, gp32 concentration dependence), and the results of these experiments were reproduced on separate days.
6
DPPH Radical Scavenging Assay
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The ability of the samples under study to scavenge the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma Aldrich Co., St. Louis, MO, USA) was measured by means of method [30 ]. An amount of 10 mL of distilled water (HLP 20UV, HYDROLAB, Straszyn, Poland) and 1.8 mL of a solution of 0.1 mM of DPPH in methanol (Sigma Aldrich Co., USA) was added to 2 g of the study sample. The obtained mixture was left in darkness for 1 h at room temperature. Then, the absorbance of the mixture was measured spectrophotometrically (UV-2600i Spectrophotometer, Shimadzu, Japan) at 517 nm, with methanol as blind feed. The calibration curve was delineated within the range from 0.1 to 100 μgmL−1 of Trolox solution (Sigma-Aldrich, Co., St. Louis, MO, USA) in ethanol. The calculation was expressed as [μmol Trolox ∗ g−1 DW].
7
ABTS Antioxidant Capacity Assay
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The antioxidant capacity was evaluated through the ABTS • + radical as described by Re et al. (1999) (link) with some modifications. A calibration curve was plotted from the standard 2.5 mM Trolox solution (Sigma-Aldrich, USA) diluted to obtain different concentrations (from 12.5 to 300 µM). For analysis, the ABTS
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