Hello community,
I am planning an ELISA for the first time. I am following a protocol from the company that sells the antigen for the standard curve (it is an antibody).
In their protocol they have the following information regarding the substrate solution:
-Add 200uL substrate solution into each well, incubate 37 degrees for 20 min
Substrate solution: 0.8uL 30%H2O2, 100uL 10mg/mLTMB in 10mL Solution A(0.05M Na2HPO4-12H2O, 0.025 M citric acid, pH 5.5)
-Stop solution: Add 50 uL 1M sulfuric acid to each well.
I've been trying to look for all the reagents I need to make all the solutions. I want to know if I use a premade TMB solution from sigma Aldrich or Thermo fisher that are "ready to use" will that make any difference? I ask because I have noticed that there are many ways to do the substrate solution (the percentage composition is different in many cases for hydrogen peroxide or the sodium phosphate salt ) and the amount per well that they suggest in their websites is 100uL, whereas in the protocol I have it says 200uL. On the other hand, I've seen that the molarity of the stop solution is 0.5M or 2M... depending on if it is a TMB from sigma or from Thermo fisher and the one in the protocol says 1M sulfuric acid.
Because of that, I am not sure which TMB should I buy... Should I buy the components individually and mix them or can I buy one of those "ready to use" TMB solutions and just keep the volumes and molarities mentioned in the protocol even though they are different from the specific instructions of the company products?
I am not sure how these things can affect my assay.
Sorry for any misspelled word or phrase in advance.
Thanks for your help
Milagros Riquelme , if you have ready to use substrate solution in you lab, you can try it. But it doesn't work always because different substrate solution ( concentration tmb) is need to different concentration sulfuric acid (0.1M - 2M) for detection. Often the concentration of TMB in this formulation and the identity of the solvent are considered proprietary information and you should not be diluted.
For example, 30% H2O2 ( contains stabilizer) https://www.sigmaaldrich.com/catalog/product/sigma/h1009?lang=en®ion=RU
And TMB isn't soluble in water and you need to dissolve it in DMSO ( or another organic solvent) and then to dilute in Solution A (and TMB dihydrochloride ) is soluble in an acetic acid)