I reacted ABTS with potassium persulfate. Within 10 min, I saw an increase in absorbance at 420 nm and after that it started to come back to baseline. I read somewhere that it is stable at least for one day.
Tjeck that the pH and iodide concentrations is as specified in the reference you read.
If I remember well, the reaction should be protected from light. By the way do you have the reference of your protocol ? That would be useful to find the problem.
Dear Mr Taheran,
You must left the solution (ABTS + K2S2O8) in the dark for 12-16 to form the ABTS+ . I think it will help you.
Sincerely,
1- Look if you do not have excess of persulfate
2- If you have organic acid in your reaction mixture (if you buffered for example)
Dear Mehrdad
because of the possibility of radical reduction by the carboxyl functional group giving rise to correponding aldehyde + peroxide.You must also pay attention to water quality you use (metals ions; use ultrapure water such as double distilled type)
It is advisable to perform the reaction in PBS buffer (pH 7.4) instead of using distilled water.
The reaction of ABTS and potassium perfulfate must take place for 12-16 hours in a covered flask under considerable stirring and protected from light exposure in order to obtain the stable ABTS·+.
After obtaining it, you must protect it from light exposure (lower to the minimum possible the lights in your lab while working with radicals). I would recommend you to split the produced radical in different tubes and freeze them. That way you could de-freeze one tube each day you perform an assay and its stability would not be compromised (always check if the same dilution gives the same absorbance at 420 nm and revise stability performing a control along with your tested samples (using solvent instead of sample and measuring absorbance at time 0 and at the end of the assay).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA