I did a little test to see if my substrate and conjugated Ig were ok.
When I added 1ul of anti Ig-Y (secondary conjugated antibody) directly to 1ml of my TMB working solution it turned orange instead of blue. Then after adding sulphuric acid it turned yellow which is fine but I was expecting it to first turn blue.
HI Catalina,
maybe you mixed up the sequence of the single steps?
if you use HRP secondary antibodies with TMB substrate, the colorless solution will become a pale blue color. after you stop the reaction with an acid it will turn yellow.
Hello Julia!
Thank you. Sorry I mixed up everything in my head. I just updated the question with the right details about what happened:
I did a little test to se if my substrate and conjugated Ig were ok.
When I added 1ul of anti Ig-Y (secondary conjugated antibody) directly to 1ml of my TMB working solution..it turned orange instead of blue...then after adding sulphuric acid it turned yellow which is fine but I was expecting to first turn blue.
Thanks!
You are right, under acetic conditions the oxidized TMB is yellow --> to measure at 492 nm. You can measure also the blue version. To stop the HRP-activity use NaN3 (but H2SO4 is easier).
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 ?
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