By the early 1950s it was known from metabolic labeling studies using radioactive phosphate that phosphate groups attached to phosphoproteins inside cells can sometimes undergo rapid exchange of new phosphate for old. In order to perform experiments that would allow isolation and characterization of the enzymes involved in attaching and removing phosphate from proteins, there was a need for convenient substrates for protein kinases and protein phosphatases. Casein has been used as a substrate since the earliest days of research on protein phosphorylation. By the late 1960s, cyclic AMP-dependent protein kinase had been purified, and most attention was centered on kinases and phosphatases that could regulate the activity of important enzymes. Casein kinase activity associated with the endoplasmic reticulum of mammary glands was first characterized in 1974, and its activity was shown to not depend on cyclic AMP.