Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro.

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).

Keywords

Cell Biology, Metabolism, Protein Biochemistry, Protein expression and purification, Signal Transduction, Cell Culture Techniques, Enzyme Assays, HEK293 Cells, Humans, Lipid Metabolism, Lipids, Phospholipids, Phosphotransferases, Transfection

Journal Title

STAR Protoc

Journal ISSN

2666-1667
2666-1667

Volume Title

2

Publisher

Elsevier

Publisher DOI

https://doi.org/10.1016/j.xpro.2021.100926

Rights and licensing

Sponsorship

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 funding

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University of Cambridge Research Outputs (Articles and Conferences)