When developing ELISA I have always followed the rule of coating just below saturation level. This avoids stacking issues. I would like to know if there are any consequences for using lower concentrations of protein to coat with? I have a belief that it can lead to variability in the assay, but I am not sure of the theory behind this and if it is correct? Coating concentration can obviously afect the range of the assay, but if coating at sub-saturation levels will it lead to problems?

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