Hi. I have some question regarding detection. If I am using HRP antibody and I will use hydrogen peroxide as substrate. What my stop solution, how much time I need to wait for the reaction, and what the nm for detection!
Hi,
it depends on chromogenic substrate you choose. Typically, tetramethyl benzidine is used for HRP/H2O2 system. Time of incubation depends on the signal, you expect (your highest OD should not be much higher than 2) - usually 15-30 min. Stop it with sulphuric acid (final conc 0.2 M) and read at 450nm.
For me I use a TMB reagent set that works perfectly and give most of time perfect results Just keep your plate at room temperature for around 20 minutes and immediately after stop the reaction by using the sulfuric acid(1M). Read at 450 nm using an ELISA Reader.
Good luck.
Best regards.
H
You can use 2M sulphuric acid (H2SO4) as stopping solution. The plates can be kept at room temperature for 20 minutes. Then you can read the optical density at 450 nm.
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