Hi Zeinab! Does the solution become yellow after a while? That could be indicative of over-oxidation over time. The blue color should be due to the TMB dimer formation after one electron oxidation (absorbance max at 370 and 652 nm). After another oxidation, it yields a molecules with absorbance at 450 nm.

In any case, the HRP/TMB assay is sensitive to several factors: H2O2 produced, concentration of TMB used, concentration of HRP employed and pH of solution (so please check also the buffer used). Here is a link to a very good source about the species formed in the assay: Article The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-...

Best regards!

Hi Dr. Luka.

Thanks for your answer. The solution becomes colorless, not yellow. I optimized all conditions (pH, H2O2 and TMB concentration,..).

"please check also the buffer used", I think this good idea and must be checked.

Thanks a lot

Best

Dear Zeinab Saberi Dehkordi,

the oxidized TMB is blue in acetate buffer pH 4, TMB is protonated in an acidic medium, and oxTMB is turned yellow if H2SO4 or any acid is added. it comes back to colorless if there are any antioxidants or reducing agents. I have seen the same situation in our lab.