I was hoping someone could solve the following:
I have an ELISA assay. Standard set up: Plate - Capture receptor - Protein (His tagged) - anti his HRP Ab
I use TMB as the substrate for the HRP. I measure the abs at 650 over 1hr.
I do not stop the reaction
The peaks reach a maximum and then decrease to 0 (formation of the 2 electron transfer diimine product).
I have been using concentration dilutions of the His-tagged protein and recording the MAXIMUM blue signal. These maxima occur at different times (it takes longer to get a maximum for lower concentrations),
My Question: Can anyone tell me if this is a problem? Should I be stopping my reaction at a certain time - I cannot see the advantage to stopping the reaction/ the disadvantage to not stopping it.
If it helps I am interested in observing a reduction in receptor - protein interaction
Any help would be great - Thanks!!