the method requires 15 microliters of sample to be incubated in 50 microliters of H2O2 for 5 minutes and 150 microliters of ammonium molybdate was added. The resulting color change was to be read using a spectrophotometer. Unfortunately, upon addition of H2O2 my samples bubbled over making it impossible to read anything. Please help with method optimization or assist
Hi, based on the Beutler method, I believe that using 100% H₂O₂ is too much. Make sure your assay is performed in quartz cuvettes, and that the final H₂O₂ concentration is around 10 mM (the working solution is usually prepared from a 30% H₂O₂ stock). To avoid bubbling and ensure reliable readings, you’ll probably need to dilute your samples. Additionally, when working with liver homogenates, it’s common to include Triton X-100 in the assay to help release the enzyme.
Beutler, E. Glutathione. In: Beutler E (ed) Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, New York, 1975; pp. 105–107.
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