OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence)

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	- Cells were incubated with 1 mM H2O2 for 30 min prior to lysis.

Protocol tips
- Cells were incubated with 1 mM H2O2 for 30 min prior to lysis.

Publication protocol

To determine channel oxidation the carbonyl groups on the protein side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) (Oxyblot, Millipore, Darmstadt, Germany). The DNP signal on RyR1 was detected by immunoblotting with an anti-DNP antibody (Millipore, Darmstadt, Germany). Protein carbonyl concentration in tissue lysates was determined using the OxiSelect Protein Carbonyl ELISA Kit (Cell Biolabs, inc., San Diego, CA). Briefly, 0.5 mg of EDL lysate was added to a 96 well protein binding plate, which was incubated overnight at 4° C. After washing the plate three times with PBS, the protein carbonyl groups were derivatized with DNPH for 45 min at room temperature (in the dark). Plates were developed with an anti-DNP antibody followed by a HRP conjugated secondary antibody. Protein carbonyl concentration was determined by comparison with a standard curve of oxidized BSA. ROS production was determined in C2C12 myotubes using the OxiSelect In Vitro ROS/RNS Assay kit (Cell Biolabs, Inc.). ROS production was measured using 0.25 mg of cell lysate according to the manufacturers recommendations. For H2O2 treated cells, cells were incubated with 1 mM H2O2 for 30 min prior to lysis. The investigators were blinded to treatment of subjects.

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