Doubts - ABTS Radical Cation Decolorization Assay

[sengwu]

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 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!

[VladimirRogovsky]

I've been working with this assay for 2 years. That's my experience:

 

1) Usually my ABTS stock solution is stable for approximately 2 weeks. I identify its degradation based on its color. If I cant obtain diluted working solution abs. 0.700 - then my stock solution is degraded. For example in first days I use 1 ml of stock solution and 99 ml of PBS to obtain abs. 0.7 in working solution, in next week I have to use 2 ml and 98 ml of PBS to obtain the same abs.

 

2) I usually prepare new diluted ABTS working solution for each experiment, because its abs. decreases significantly even during the experiment.

 

3) Usually SEM (standard error of the mean) in my ABTS experiments is more, than 0.05, so for me its no difference whether the range of abs. is 0.700±0.02 or 0.700±0.05.

 

4) I usually use 2.45 mM potassium persulfate and 7 mM ABTS in a ratio of 1:1, so I have no experience of mixing two times concentration of potassium persulfate and ABTS.

 

5) I have the same experience. Abs. of ABTS working solution is not stable and decreases even during experiment. I dont use 96-well plate, instead of this I use single plate. After 3-5 experimental tests I determine abs. of control ABTS working solution. Usually I incubate my probes for 2 minutes. When I analyse my results I compare each experimental result with its own control and determine the percent of abs. decreasing.

Any way exposure to light of stock and working solution should be minimized.

 

I'm glad if my experience can help you.