When I add TMB to my HRP substrate it changes to blue, dependence on the concentration of HRP this blue color is dark or light. After I add HCL 2 M as stop reaction, the solution becomes yellow. But after 2-3 min the intensity of yellow color decreases dramatically.
Does anyone have any similar experience? TMB was bought 2 years ago, but it always kept in 4-8 C
Should I measure OD of HRP/TMB after adding stop solution immediately?
Dear Hoda
You should measure the colour intensity within about 1 hour after adding the stop solution. The colour should be stable for this time. If not you should check the concentration of the acid stop solution. I seem to remember that a 1M HCl or 0.6 M H2SO4 solution is used and your acid concentration may be too high. The acid is used to stop the reaction between the H2O2 and the TMB which is catalysed by the HRP
The fact that you are seeing the dark blue colour change indicates that your reagents are still good
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