The quantity of frataxin captured in each well is proportional to the amount of horseradish peroxidase activity within each well. The quantity is the change in absorbance at 600 nm/minute/amount of sample loaded into the well. Examine the linear rate of increase in absorbance at 600 nm with time. As shown, the rate/slope is calculated between these time points. For the control or normal sample, the rate versus amount loaded is plotted as a straight line in the linear region of the assay as shown. Compare the rates of the control (normal) sample and with the rate of the null (background) and with your unknowns, experimental or treated samples to get the relative amount of frataxin.
Abcams' protein quantity microplate assays use the well-established sandwich ELISA format, whereby capture and detector antibodies are used to immobilize and then quantify a target protein or enzyme. All of our microplate assays utilize our highly-validated immunocapture antibodies, which are able to capture large, multi-subunit enzyme complexes in their fully intact state. Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, a second monoclonal antibody, against a different epitope on the target, is added to the well. This detector antibody is either directly labeled with biotin, or a biotin-labeled goat anti-mouse secondary is added. Substrate plus HRP or AP conjugated to streptavidin provide a colorimetric signal that is readable by any plate readers capable of standard ELISA absorbance measurements.