Hi dear researchers
We used HRP (Biobasic) in phosphate buffer (pH: 6.5) and after adding TMB, the reaction turning to light blue without adding H2O2. After adding H2O2 to reaction media, the brown color was produced (not expected blue color).
There were no changes after decreasing TMB, HRP and H2O2 concentration.
We expect that after adding TMB to the enzyme without H2O2, the reaction doesn’t start and after adding H2O2, we expect that see blue color.
( I have checked and rechecked all calculations, buffer composition, pH etc. )
That's alright. You have mentioned virtually everything you did but what happened to the addition of SDS to promote the stabilization of the blue colour? It might be what is missing. Please see the attached document: Article A novel efficient medium for chromogenic catalysis of tetram...
Just curious! Can you add TMB and H2O2 together instead of one after another?
Is it turning yellow after you stop the reaction with acid?
There is information on the reaction of, and colours generated by, HRP , TMB and H2O2 on Wickipedia. Use search terms HRP, TMB, H2O2.
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