The regulation of lipid kinases has remained elusive given the difficulties of assessing changes in lipid levels. Here, we describe the isolation of protein and lipid kinases to determine the regulation of lipid kinases in vitro. This can be followed by analysis of effects of regulators on lipid kinase-mediated changes in phospholipids without the use of radioactivity, with a specific focus on PI(5)P generation by the enzyme PIKfyve. For complete details on the use and execution of this protocol, please refer to Karabiyik et al. (2021).We are grateful for funding from the UK Dementia Research Institute (funded by the MRC, Alzheimer’s
Research UK and the Alzheimer’s Society) (to DCR), Roger de Spoelberch Foundation (DCR) and the
Gates Cambridge Scholarship (CK) for fundin
