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After a second wash, the 3,3',5,5' Tetramethylbenzidine substrate solution (Biovendor, Candler, NC) was applied for 10 min, color development was stopped with an acidic stop solution, and the optical density was measured at 450 nm using a plate reader.
TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution.
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The blue reaction was changed to yellow by an addition of 50 μL acidic stopping solution.
An acidic stopping solution was then added, and the enzymatic turnover rate of the substrate was determined by dual wavelength absorbance measured at 450 and 620 nm.
After a third incubation and washing step, the substrate, tetramethylbenzidine was added and incubated briefly before adding an acidic stopping solution.
After addition of acidic stopping solution (EUROIMMUN), the optical density (OD) was read at 450 nm using an automated spectrophotometer (Spectra Mini; Tecan, Crailsheim, Germany).
An acidic stopping solution was then added, and the degree of enzymatic turnover of the substrate was determined by a dual-wavelength absorbance measurement at 450 and 620 nm.
If you've been overdoing the acidic fertilising, stop now!
Previously, we showed that a pH shift to the acidic range stopped 1,3-PDO production and increased the butanol/1,3-PDO ratio significantly (data not shown).
The reaction was stopped by adding an equal volume of acidic (2N HCl) stop solution.
But heat built up on roofs by the sun's rays - at temperatures as low as 150 degrees Fahrenheit - also sets off the fire-stopping acidic reaction that the wood's designers intended to happen only at the temperatures of an actual house fire, about 400 degrees.
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