Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

  Functional Studies - Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

Abcams' enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states. 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, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.

  Sandwich ELISA - Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

Abcam's 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.

  - Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

Figure 1. To determine the activity in the sample, calculate the slope by using microplate software or by manual calculations using one of the two methods shown. Compare the sample rate with the rate of the control (normal) sample and with the rate of the null (background) to get the relative Complex IV activity. (A)The rate is determined by calculating the gradient of the initial slope over the linear region. (B) The rate is determined by calculating the slope between two points within the linear region.

  - Complex IV Human Specific Activity Microplate Assay Kit (ab109910)

Figure 2. The quantity of Complex IV is expressed as the amount relative to a normal or control sample. Examine the color development and ensure that the rates are linear as shown in the graph. Subtract the initial absorbance reading from the final absorbance reading to determine the relative quantity of Complex IV captured in each well.