FAQ: Hydrogen Peroxide and Peroxidase Assays

Q: What is the difference between your Hydrogen Peroxide Assays #STA-343 and #STA-344?

A: The difference between these two assays is the detection method.  The Hydrogen Peroxide Assay #STA-343 uses a colorimetric plate-based format and has a sensitivity of detection of approximately 1 µM hydrogen peroxide.  The Hydrogen Peroxide/Peroxidase Assay #STA-344 uses a fluorescence plate-based format and has a limit of detection of 50 nM hydrogen peroxide.  We recommend #STA-344 if you have access to a fluorescent plate reader because of its superior sensitivity. 

 

Q: Can these kits be used on frozen samples?

A: Both assays #STA-343 and #STA-344 can be used on frozen samples, but due to the transient nature of ROS samples should be frozen for no longer than 1-2 months at -80ºC.  We don’t recommend storing samples for this long, if possible, since the best results will come from fresh samples.

 

Q: Are cell and tissue samples compatible with these assays?

A: Cell and tissue lysates are compatible with our Hydrogen Peroxide/Peroxidase Assay #STA-344 but not with our colorimetric assay #STA-343.  Tissue lysates can be prepared by resuspending tissues in 1X PBS or 1X Assay Buffer, followed by homogenization or sonication, centrifuge at 12000 xg for 10 min, and harvest the supernatant as the lysate.

 

Q: Will phenol red interfere with these assays?

A: Both colorimetric and fluorometric assays are compatible with cell culture supernatant samples and phenol red will not interfere with either assay.  A blank well consisting of just the media should be included as negative control.

 

Q: Will EDTA interfere with these assays?

A: The presence of EDTA in plasma samples should not interfere with our colorimetric assay as long as the concentration of EDTA is below 200 µM.  Above this concentration the assay results will not be reliable.  If samples have EDTA concentrations above this level, you may want to consider using our fluorometric format.  This assay uses a different chemistry and is therefore compatible with higher EDTA concentrations.

 

Q: Are these assays compatible with DTT or ß-mercaptoethanol?

A: Reducing agents such as DTT and ß-mercaptoethanol will interfere with our fluorometric Hydrogen Peroxide/Peroxidase assay, but they are compatible with the colorimetric format.

 

Q: Will hydrogen peroxide degrade during cell lysis

A: ROS in general is not very stable, but hydrogen peroxide is more stable relative to other ROS, so degradation is not typically a concern.  The bigger concern is with introducing ROS during cell lysis, which is impossible to avoid. However, any changes in ROS due to sample preparation should be uniform across all samples if they are prepared under the same conditions and at the same time.  An alternative is to use our Intracellular ROS Assay kit #STA-342 which measures ROS of cultured cells and does not involve making a cell lysate.  Hydrogen peroxide makes up the majority of ROS measured with this kit.

 

Q: Is it recommended to test for damage markers prior to testing for hydrogen peroxide?

A: Most researchers studying oxidative stress measure multiple markers and once they confirm detection of a marker they often test ROS directly.

 

Q: Once opened, does the entire kit need be used at that time?

A: No, the kit can be used over multiple experiments.  The working reagents are only stable for one day, so these will have to be prepared in a volume that is sufficient for the number of reactions being performed each time.  The standard curve dilutions should also be prepared each time the assay is run.