Hello all,
I am trying to standardise the protocol for the above test, am unable to get results properly. For this method I used, 200mcl of different concentartion + 1ml of PBS of pH 7.4 + 160 mcl of ABTS reagent ( ABTS 2mM with 17mM POTASSIUM PERSULPHATE, prepared freshly with 16 hr incubation). Later all these were incubated in dark at room temperature for 20 minutes and read at 630 O.D. kindly help.
Hi
Amulya
The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 14 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.01 units at 734 nm using a spectrophotometer. Fresh ABTS solution was prepared for each assay. Plant extracts (1 ml) were allowed to react with 1 ml of the ABTS solution and the absorbance was taken at 734 nm after 7 min using a spectrophotometer. The ABTS scavenging capacity of the extract was compared with that of BHT and ascorbic acid and percentage inhibition calculated as ABTS radical scavenging activity (%)=Abscontrol−AbssampleAbscontrol where Abscontrol is the absorbance of ABTS radical in methanol; Abssample is the absorbance of ABTS radical solution mixed with sample extract/standard. All determinations were performed in triplicate (n = 3).
Arnao MB, Cano A, Acosta M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 2001;73:239–44.
ABTS stock solution of 7mM with 2.45mM potassium persulfate1/1 V/V first to be prepared and left for 4 hr until the reaction completed as shown by stable absorbance.then diluted with ethanol to read absorbance of 0.700 at734nm.The assay then conducted on 0.9 ml of ABTS solution and 0.1 ml of your sample mixed well measured absorbance immediately at 734nm using a spectrophotometer .good luck
Note of caution on the method: ABTS radical is not a free radical that is generated in organisms such as hydroxyl and superoxide radicals. Therefore, estimation of total antioxidant status against an artificial radical such as ABTS (or any other not natural radical) is just an indication of the antioxidant potential of your sample, and not an accurate estimation.
How about if I use ammonium persulfate instead of using Potassium persulfate? is it okay?
Amulya .G
for ABTS assay
which dilution of ABTS SOLUTION you are using to get 0.746 at 734nm
thank you
Kais Kassim Ghaima what do I mix my potassium persulfate with to make the potassium sulfate solution?
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