Am doing a pilot run of Sandwish ELISA using the R&D Systems DuoSet ELISA kit. There's no substrate provided in this kit, so I use one from BD. I prepare the sustrate 15mins before addition to the wells by mixing Reagent A-TMB and Reagent B-Hydrogen peroxide in a ratio of 1:1.
All reagents are left to attain room temperature before starting the assay
Why is there no blue color development after adding TMB substrate solution?
Thanks
Hi Rhoda, it is sometimes a problem trying to combine kits that might not be compatible.
Plus the companies will not specifically tell you what is in their kits.
My feeling is that there might be NaN3 (azide) in one of the reagents. Azide totally kills HRP.
I would use a total kit with everything included.
You can check the compatibility of the reagents by a simple test:
Take the leftovers of your substrate mixture and add 1 µL of the labeled secondary antibody dilution. The solution should turn to blue (or even greenish) immediately. If there is no color, something went completely wrong.
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