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Doubts - ABTS Radical Cation Decolorization Assay


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#1 Seng

Seng

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Posted 03 June 2011 - 10:28 PM

Dear all,

Iím currently learning an antioxidant assay called ABTS scavenging capacity. Iíve 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 itís degraded based on its color and etc.?

2) How many days does the diluted ABTS working solution (diluted with MeOH to obtain abs. 0.700Ī0.02) can stable for? How can I identify whether itís 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 0.700Ī0.02? Can it be in the range of 0.700Ī0.05 (I found one journal calibrate to get this abs)?

4) Iíve 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 didnít dissolve well.

5) Another problem that Iím super concerned is the stability of absorbance of ABTS stock solution while preparing diluted ABTS working solution. How come when I calibrate to abs. 0.700Ī0.02, 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!




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