Oxidative Stress: OxyBlot Protein Oxidation Detection Kit (S7150)

Oxyblot (Millipore) was performed on the ischemic and nonischemic myocardial tissue as per manufacturer protocol. Briefly, tissue was lysed in radioimmunoprecipitation assay buffer (Boston BioProducts) and 2 aliquots of each specimen were analyzed (1 aliquot was subjected to a derivatization reaction, while the other aliquot served as a negative control). A 5‐μL protein sample derived from each experimental animal was divided into 2 eppendorf tubes and then denatured with 12% sodium dodecyl sulfate. For the positive control, 2,4‐dinitrophenylhydrazine solution was used to derivatize the samples. Derivatization control solution was used in the control samples. Both sets of samples were incubated at room temperature for 15 minutes after which neutralization solution was added. Finally, the 2 samples were loaded onto a polyacrylamide gel and followed by SDS‐PAGE, Western blot transfer, and immunodetection.