Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit - FAQs

View additional product information for Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit - FAQs (A22188)

3 product FAQs found

I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?

The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H2O2 oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can I use the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Cat. No. A22188) to measure H2O2 from frozen cell culture supernatant?

We recommend using fresh samples that have not been frozen since H2O2 degrades over time and more readily in diluted form or in solutions that contain metal ions or natural antioxidants. For assaying cell culture supernatant, we would recommend analyzing samples as soon as possible.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can Amplex Red Assays be performed using cell lysates?

This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H2O2 required for the reaction, competing with HRP (and catalase) for the substrate.

The Amplex Red Assays are best performed with either purified enzymes or extracted H2O2 in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.