Stop reagents have to fulfil two major requirements: 1) to terminate the reaction by effectively inhibiting the enzymatic activity of HRP, 2) to stabilize the oxidized products of the chromogenic or fluorogenic substrate(s).
Strong (mineral) acids are used as stop reagents to inhibit peroxidase enzymatic activity. This is especially the case when 3,3′-5,5′ tetramethylbenzidine (TMB) serves as a color substrate and the peroxidase enzyme is inhibited by adjusting the pH to values of pH 2 or even lower.
In the case of TMB, the stop solution, sulfiric acid, not only inhibits color development but also converts the blue oxidation product of TMB to a yellow derivative which has a significantly higher molar absorptivity al 450mn.
The concentration of H2SO4 to be used, depends on the manufacturer, and for same the chromogenic substrate, they propose different [H2SO4].
-XXX TMB Substrate: Recommended stop solution is 0.18 M sulphuric acid.
- ZZZ : 2M sulfuric acid
- even some manufactures do not specify, i.e., YYY: “Upon acidification (to stop the enzymatic reaction) the reaction product turns yellow with an absorbance peak at 450 nm.”
I´d like to know how it can, the different concentrations of acid used, affect ? what it is difference between using 0.2M or 2M? not only regarding the hazardness of the solution, but in terms of stabilization of oxidize products. Does the more concentrated stabilized better?
Lourdes,
it depends on the buffering capacity of the chromogenic solution. In my experience, stopping 100ul chromogen with 100ul 1M H2SO4 works fine. If you're really curious, stop mixtures of chromogen (maybe you have some leftovers) and a little diluted peroxidase with various concentrations of acid and measure both at 405nm (or ideally 650 or 370) and 450nm to see how well the resulting dye is protonated. If you have a spectrophotometer, record UV/Vis spectra of the stopped solutions for best results.
Lourdes,
it depends on the buffering capacity of the chromogenic solution. In my experience, stopping 100ul chromogen with 100ul 1M H2SO4 works fine. If you're really curious, stop mixtures of chromogen (maybe you have some leftovers) and a little diluted peroxidase with various concentrations of acid and measure both at 405nm (or ideally 650 or 370) and 450nm to see how well the resulting dye is protonated. If you have a spectrophotometer, record UV/Vis spectra of the stopped solutions for best results.
I agree with Wolfgang.
Since sulfuric acid is a very strong acid, the total amount of acid is most important.. You might titrate your blue TMB solution until no change in UV/VIS spectrum is detectable (perhaps you can see it by eye). In some assays the volumes of the substrate and the stop solution are not the same. This has to be considered, too. I do not think that higher acid concentrations stabilize better - however, I've never seen an experimental study about this question.
1N or 2N HCl can be used as stop solution for TMB instead of 2M H2SO4
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