Cardiac troponin I phosphorylation has been shown to be reduced in human heart failure and hypertrophic cardiomyopathy using the increasingly popular Pro-Q Diamond phosphoprotein gel stain. In this brief report I discuss the use of Western immunoblotting, non-equilibrium isoelectric focussing and Pro-Q Diamond and introduce phosphate affinity SDS-PAGE using Phos-tag-acrylamide for the investigation of troponin I phosphorylation.