Phosphatidylinositol 4-kinase and phosphatidylinositol 4-phosphate 5-kinase assays

Inositol lipid kinases are perhaps the easiest and most straightforward enzymes in the phosphoinositide pathway to analyze. In addition to monitoring lipid kinase-specific activity, lipid kinase assays can be used to quantify the inositol lipids present in isolated membranes (Jones et al., Methods Mol Biol 462:75-88, 2009). The lipid kinase assays are based on the fact that the more negatively charged phosphorylated lipid products are readily separated from their lipid substrates by thin layer chromatography. We have summarized our current protocols and identified important considerations for working with inositol lipids including different methods for substrate delivery when using recombinant proteins.