Hello all.
We are trying to prepare TMB (HRP substrate) in house. 1 mg TMB (biobasic) was dissolved in 10 mL phosphate-citrate buffer (pH 5.0). Two uL H2O2 was added and an aliquot of complete reagent was mixed with HRP. The reaction produced orange color and not expected blue color. Upon adding H2SO4, it turned to yellow color. Reagent without H2O2 did not produce any color upon mixing with HRP. Please see attached photos.
Why we are not getting blue color? I have checked and rechecked all calculations, buffer composition, pH etc.
Sincerely,
Syed
Dear Syed A Ali,
İ sent you interesting article.
https://www.thermofisher.com/az/en/home/life-science/protein-biology/protein-assays-analysis/elisa/elisa-enzyme-substrates-selection-guide.html
http://www.interchim.fr/ft/B/BA357a.pdf
Regards, Shafagat
I couldn't figure out. Looks like commercially available TMB reagents contain certain proprietary reagent(s).
Hey Syed,
I have recently experinced the same thing. Have you figure it out? In my case, I used the commercial TMB assay kit from KPL where TMB substrate and H2O2 are just provided. Looking forward to hearing from you! Thanks.
Even I was facing the same problem. When I added TMB substrate to IgG-HRP it turned blue for a sec and after that the blue color turned orange. Can anyone explain the possible reason ?
Hello colleagues
I think it is because of the second oxidation step forming diamine
For TMB
First oxidation (charge transfer intermediate looks blue)
second oxidation (diamine looks yellow)
Please decrease your concentrations (HRP 10 ng) and (H2O2 5 to 10 mM)
Add SDS prior to oxidation, the negative charge will stabilize the blue color.
You will need to optimize the conditions first.
Prepare TMB (40 mM) stock in DMSO. Use working concentration of 1 mM in buffer.
Use low concentration of H2O2 (1 to 10 mM).
Use HRP concentration of 10 ng per reaction.
Then you can see see the color transition blue, green and finally yellow
Simply dilute it. It is because of overoxidation. reduce the amount of HRP
Yes, as Sharma recommend, dilution of the compounds will avoid the effect of overoxidation. Less peroxide, less TMB and less HRP. Imagine: In an ELISA you have not very much enzyme bond via the antibodies onto the well surface. Substrate solutions contain usually around 0.5- 1 mM TMB and 5-10 mM H2O2.
Thank you! I was just having the same problem, when dot-blotting to check the secondary AB. But this seems to be just to much HRP, then.
Hello there, I am facing a similar issue. I am getting blue color only in certain enzyme substrate combinations. Keeping the enzyme concentration constant, when TMB conc is decreased, I get brown colored complex instead of blue. And on the other hand when I keep the TMB conc constant and decrease the HRP concentration, I get brown coloured complexes at lower conc of HRP. Can you please suggest the reason for this? Thanking you
Devanshi S. Shah im getting the same results as you. Hoping someone can help well!
Its because of the overoxidation of TMB due to the presence of high concentrations of HRP and hydrogen peroxide. Try to use a fixed concentration of HRP then use various ratios from (5-20 uL). maintain the pH of the buffers (4 for the TMB and 7-7.4 for HRP). I hope this could help you to overcome this issue.
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