Q: How does the His-Tag Protein ELISA work?
A: Our His-Tag Protein ELISA (AKR-130) is a competitive ELISA that generates a reverse curve, with the lowest OD values obtained from high His concentrations. First, the sample is added to a plate coated with polyhistidine. The antibody is added after the sample, which competes for His in the sample and on the plate. Samples with high His concentrations will bind more antibody, which leaves less available to bind to the polyhistidine conjugate on the plate and therefore a lower signal.
A: Our ELISA will recognize both N- and C-terminal His-Tag fusion proteins.
Q: What sample dilution should I use?
A: The amount of sample to add to the assay will depend on the level of His in the sample. Our recommendation is to perform a sample titration against the standard curve to determine the optimal dilution necessary so that the samples fall within the middle of the standard curve.
Q: How do I calculate the His-tagged protein concentration of my unknown samples?
A: The best way to determine concentrations from a standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel. The standard curves generated with ELISAs are not typically linear, therefore the points of the curve that aren’t in the linear portion should be eliminated, so that you get r2 as close to 1 as possible, as long as your samples still fall in that range. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is: (OD value-b)/m for linear lines or ln(x)=(OD value-b)/m for logarithmic lines.