These preparation protocols are intended as a guide for preparing unknown samples. The user may need to adjust the sample treatment accordingly. All samples should be assayed immediately or stored for up to 2 months at -80 °C. A trial assay with a representative test sample should be performed to determine the sample compatibility with the dynamic range of the standard curve. High levels of 4 interfering substances may cause variations in results. Samples may be diluted in 1X Assay Buffer as necessary before testing. Run proper controls as necessary. Always run a standard curve with samples. Note: Please see the Potential Interference and Compatibility section for chemicals that may interfere with assay results. • Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C. • Plasma: Collect blood sample and add to a blood collection tube containing heparin as the anticoagulant. Centrifuge at 3,000 rpm for 10-15 minutes at 4 °C. Remove the upper yellow plasma supernatant layer without disturbing the white buffy coat (leukocytes). Samples should be tested immediately or frozen at -80 °C. Note: Hemolyzed plasma or serum should be avoided. Heparinized plasma is recommended over EDTA plasma. • Cell lysates: Lyse 1-2 x 106 cells/mL by sonication or multiple freeze-thaw cycles in 4 volumes of cold PBS or 1X Assay Buffer. Centrifuge at 12,000 rpm for 15 minutes at 4 °C and remove insoluble cell material. A high concentration of protein may interfere with the assay. In this case, filter the sample with a 10 kDa MWCO centrifugal filter before assaying (to reduce protein interference and turbidity). Test samples immediately or store at -80 °C. • Tissue lysates/homogenates: Homogenize/sonicate approximately 10 mg of tissue in 1-2 mL cold 1X Assay Buffer. Centrifuge the homogenate at 12,000 rpm for 15 minutes at 4 °C and collect the supernatant. A high concentration of protein may interfere with the assay. In this case, filter the sample with a 10 kDa MWCO centrifugal filter before assaying (to reduce protein interference and turbidity). Test samples immediately or store at -80 °C. • Urine: Test neat or diluted with PBS or 1X Assay Buffer if appropriate. • Lipophilic Fractions: Dissolve lipophilic samples in 100 % ethanol or acetone and then dilute in 100 % ethanol or 50 % acetone. Incubate the mixture for 1 hour at room temperature with mixing. Further dilute samples as necessary prior to testing. • Food Samples: Results may vary depending on sample source and purification. Dilution and preparation of these samples is at the discretion of the user, but use the following guidelines: o Solid or High Protein Samples: Weigh solid sample and then homogenize after adding deionized water (1:2, w/v). Centrifuge the homogenate at 10,000 x g for 10 minutes at 4 °C. Recover the supernatant which is the water-soluble fraction. The insoluble fraction (pulp) is further extracted by adding acetone (1:4, w(solid pulp)/v) and mixing at room temperature for 30-60 minutes. Centrifuge the extract/solid at 10,000 x g for 10 minutes at 4 °C. Recover the acetone extract and dilute with PBS, 1X Assay Buffer, ethanol or water as necessary prior to running the assay. The TEAC value is calculated by combining the results from the water- soluble fraction and the acetone extract from the pulp fraction. o Aqueous Samples: Centrifuge the sample at 10,000 x g for 10 minutes at 4 °C to remove any particulates. Dilute the supernatant in PBS or 1X Assay Buffer as necessary prior to running the assay. 5 Potential Interference and Compatibility Table 1 contains some common substance compatibilities. The listed concentration values are prior to performing the assay. Dilution of the substance/buffer, and ultimately samples, may be required to completely eliminate interference. Even with some interference, accurate quantitation can be achieved by running standards in the same buffer as samples, although kit sensitivity may be compromised. Substance Compatible Concentration Acetone 50 % Deoxycholic acid 0.5 % Dithiothreitol Not Compatible DMSO 5 % EDTA Not Compatible Glycerol 50 % 100 mM Hepes Not Compatible Methanol 50 % 2-Mercaptoethanol Not Compatible NP-40 Not Compatible PBS undiluted SDS Not Compatible Tris, pH 7.5 500 mM Triton-X 100 0.1 % Tween-20 Not Compatible Table
- Substance Compatibilities
Calculation of Results
- Calculate the average absorbance values for every standard, control, and sample.
- Plot the average absorbance for the standards against the final concentration of the trolox standards from Table 2 (hydrophilic assays), or Table 3 (lipophilic assays) to determine the best curve. See Figures 2 and 3 for example standard curves.
- Determine the antioxidant concentration, as μM Trolox equivalents (TEAC value), in of the samples with the equation obtained from the linear regression analysis of the standard curve. Substitute the average absorbance values for each sample. Remember to account for dilution factors. Only use values within the range of the standard curve.
- (Sample average absorbance - (y-intercept)) Antioxidant (μM) = x Sample dilution Slope Results may also be expressed as micromoles of Trolox equivalents per gram of sample (TE μmol / g). The formula weight of Trolox is 250.29 g / mol.