May 18, 2026
Measuring catalytic oxygen reduction by purified proteins using a Seahorse XFe96/XF96 Pro Analyzer
- Erik Lacko1
- 1University of Glasgow
Protocol Citation: Erik Lacko 2026. Measuring catalytic oxygen reduction by purified proteins using a Seahorse XFe96/XF96 Pro Analyzer. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyob1qgx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: April 10, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 314823
Keywords: oxygen reduction, Seahorse XF technology, purified proteins, high-throughput, purified redox enzyme, redox enzyme, measuring catalytic oxygen reduction, measuring oxygen consumption, record oxygen levels during the first measurement cycle, record oxygen level, catalytic oxygen reduction, reproducible readout of enzymatic activity, dissolved oxygen, oxygen consumption, xf96 pro analyzer, purified protein, xf96 pro analyzer this protocol, enzymatic activity, seahorse xfe96, interactions between purified protein, assay
Abstract
This protocol describes a microplate‑based method for measuring oxygen consumption by purified redox enzymes using the Agilent‱ Seahorse XF Analyzer. The workflow outlines how to prepare reagents, load injection ports, initiate reactions, and record oxygen levels during the first measurement cycle after injection.
The method enables users to quantify catalytic oxygen reduction in vitro or to detect redox‑dependent interactions between purified proteins. When applied correctly, the assay yields a clear, concentration‑dependent decrease in dissolved oxygen following reaction initiation, providing a reproducible readout of enzymatic activity.
Materials
Agilent‱ Seahorse XFe96/XF Pro Analyzer
Agilent‱ Seahorse XFe96/XF Pro sensor cartridge (Agilent, Cat no. 103792-100)
Agilent‱ Seahorse Calibrant solution (Agilent, Cat no. 100840-000)
Agilent‱ Seahorse XF96 microplate (Agilent, Cat no. 103794-100)
Safety warnings
Monitor and export Oxygen level as opposed to the default OCR
Before start
Hydrate cartridge the day before or set desired instrument operating temperature.
Day before the experiment
Hydrate a Seahorse XFe96/XF 96 Pro sensor cartidge using a XF calibrant solution according to manufacturers instructions and incubate overnight in the dark. Turn on the instrument and set to the desired operating temperature.
Experiment day
Set up assay protocol:
Mix/Measure cycles: minimum duration available, typically 2 minutes on modern instruments
Include at least 3 baseline measurement cycles before injecting port A
Include at least 3 post injection measurement cycles
Load drug port A on the sensor cartridge with 10 uL of reducing agent/reaction initiator at 10X the active concentration
Perform cartridge calibration step
Load 90 uL of purified enzyme onto a Seahorse XFe96/XF 96 pro microplate wells, avoid loading the four corner wells that are used for background subtraction.
Once calibration is finished, load the microplate to begin measurements
After the run has finished , export per well data set to Oxygen level instead of OCR. Quantify average oxygen level across the last measurement cycle in the baseline prior to injection, and quantify the average oxygen level across the first measurement cycle after injection. Quantify oxygen reduction by: Oxygen reduction = Oxygen level before injection - Oxygen level post injection