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Reagents Preparation: 1. Add exactly 3.5 mL of ultrapure water in each vial of Reagent B and mix thoroughly. Once dissolved, keep refrigerated at -20 °C. (solution B). 2. Prepare FRAP working solution just before use by mixing Reagents A, B and C (10:1:1). 3. FRAP standard: Add exactly 1 mL of ultrapure water in each Standard vial and mix thoroughly. Prepare standards immediately prior to the assay performed. Do not store the standard preparations.
Sample Preparation: Dilute your sample to an absorbance value corresponding to 500-600 μM of standard approximately. Plasma samples do not usually need to be diluted.
Standard Preparation Antioxidant activity is expressed as FRAP values (Ferric Reducing Ability of Plasma). These values are related to Fe2+ concentration. Prepare calibration curve in 1 mL tubes as shown below in Table 1 (see kit booklet).Performing the assay: 1. Add 10 μL of the sample or standard in each well. 2.Add 220 μL of FRAP working solution previously prepared (see Reagents Preparation) in each well. Final dilution of the sample in the reaction mixture will be therefore, 1/22. 3.Mix the mixture for 4 minutes under continuous stirring. 4.Read the absorbance at 593 nm.
1.Zeroed the absorbance values: ?A593nm = ?A593nm sample/standard - A593nm blank. Where A593nm sample/standard is the absorbance measured 4 minutes after the addition of antioxidants from samples or standards. 2.Plot the zeroed absorbance (?A593nm) of standards as a function of their final concentrations (Table 1). See Figure 2 for a typical standard curve (see images). 3.Calculate the FRAP value of the samples using the equation obtained from the linear regression of the standard curve substituted ?A593nmvalues for each sample. FRAP (μM) = (?A593nm- intercept)/ slope