Doubts - ABTS Radical Cation Decolorization Assay - (Jun/04/2011 )

Dear all,

I’m currently learning an antioxidant assay called ABTS scavenging capacity. I’ve got several questions intend to ask, as follows:

1) How many days does the ABTS stock solution (2.45 mM potassium persulfate and 7 mM ABTS salt solution in a ratio of 1:1 v/v) can stable for? How can I identify whether it’s degraded based on its color and etc.?

2) How many days does the diluted ABTS working solution (diluted with MeOH to obtain abs. 0.700±0.02) can stable for? How can I identify whether it’s degraded based on its color and etc.? According to Re et al. (1999), the diluted ABTS working solution can be stabled for more than 2 days.

3) Is it the range of abs. has to be in 0.700±0.02? Can it be in the range of 0.700±0.05 (I found one journal calibrate to get this abs)?

4) I’ve got prepared the ABTS stock solution according to one journal by mixing two times concentration of potassium persulfate and ABTS in a ratio of 1:1 v/v (4.88 mM potassium persulfate and 14 mM ABTS. After incubating in the dark for 12-16 h, it became grey color instead of dark green. So, is it considered to be degraded? I had tried to dilute this grey color stock with MeOH but it didn’t dissolve well.

5) Another problem that I’m super concerned is the stability of absorbance of ABTS stock solution while preparing diluted ABTS working solution. How come when I calibrate to abs. 0.700±0.02, the 1st min I obtained 0.710 but after 5 min it decreased to 0.679. For your info, I perform ABTS assay using 96-well plate and I calibrate under direct light source in the room. Is it the decrement of abs. because of high sensitivity of ABTS to light? Should I perform in a place with minimum light source? However, I assume ABTS stock solution supposes to be stabled after 12-16 h incubation in the dark at room temp.

Do hope you guys could provide good answer to my questions especially question no. 5.

Thanks!

Try this one

TEAC microplate assay: place in microplate wells 250 µl of assay buffer, and then add 15 µl of standard or sample ) or dH2O (blank). Read the initial absorbance at 660 nm for the first absorbance point. Add 37.5 µl of ABTS Radical Solution to wells and incubate 10 min at room temperature or 5 min at 37 °C. Read the absorbance a second time at 660 nm.

End point scanning of the reaction products is done after reducing remaining ABTS radicals by adding NaN3 to a final concentration of 60 mM. 

Reagents

Sodium acetate buffer 0.4  M at pH 5.8 (Assay buffer): mix 940 ml sodium acetate 0.4 M (dissolve 32.8 g sodium acetate anhydrous in 1 liter dH2O) with 60 ml glacial acetic acid 0.4 M (dilute 22.8 ml glacial acetic acid with 977.2 ml of dH2O), and store at 4 °C, resulting stable 6 months.

Sodium acetate buffer 30 mM at pH 3.6 (ABTS buffer): mix 75 ml sodium acetate 30 mM (dissolve 2.46 g sodium acetate anhydrous in 1 liter dH2O) with 925 ml glacial acetic acid 30 mM (dilute 1.71 ml glacial acetic acid with 998,28 ml of dH2O), and store at 4 °C.

ABTS reagent: dissolve 0.549 mg (10 mM) of ABTS in 100 ml ABTS buffer, and store protect by light, wrapping with aluminium foil, at 4 °C.

ABTS radical solution: dispense 27.8 µl (2 mM) of peridrole 37% in 100 ml of ABTS reagent and incubated for 1 h at room temperature so that bluish-green color appeared and then it was stored protect by light, wrapping with aluminium foil, at 4 ˚C, resulting stable 6 months.

For further information contact to me: francesco_zagami@virgilio.it