Measurement of phosphoinositides in the zebrafish Danio rerio

David R. Jones, Irene Barinaga Rementeria Ramirez, Martin Lowe, Nullin Divecha

Research output: Contribution to journalArticlepeer-review


Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [32P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [32P]γATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days. © 2013 Nature America, Inc. All rights reserved.
Original languageEnglish
Pages (from-to)1058-1072
Number of pages14
JournalNature protocols
Issue number6
Publication statusPublished - Jun 2013


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