I am looking for a way to detect Phosphatidylinositol (3,4,5)-trisphosphate levels in hearts of mice. All I can find are methods that employ cultured cells. Is there a reliable method to extract and measure PIP3 levels from frozen tissue?
Not aware of any good method either. I can imagine that this is a challenging task. You can do lipid extraction from the tissue using documented methods for glycerophospholipid extraction. However, the major difficulty lies in the analysis of assumingly minute quantities of PIP3 from the milieu of the complex extracted lipid matrix. My best guess for this analysis is to go with a high resolution mass spectrometer. Recent studies on lipidomics using mass spectrometry may help you identify a viable MS method to try, (mostly cellular studies as you already have identified). You may be a pioneer in trying to accomplish the detection of PIP3 in tissue!
Just thinking loud, If only we had a fluorescent molecule that we could use as a probe that would binding to PIP3 (or just PI'S) on tissue or the extracted lipids from tissue, then we may be able use capillary electrophoresis with LIF.
The following method may be tried for reproducible results
J Biol Chem. 1997 Feb 28;272(9):5477-81.
A novel, rapid, and highly sensitive mass assay for phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and its application to measure insulin-stimulated PtdIns(3,4,5)P3 production in rat skeletal muscle in vivo.
van der Kaay J1, Batty IH, Cross DA, Watt PW, Downes CP.
Http://www.readcube.com/articles/10.3390/metabo2010057?locale=en
maybe this helps you,
Using mass spectrometer with specific scan for PIP3 suh as MRM!, NL, however, you may need the specific extraction protocol for PIP species.
..and after the proper extraction protocol to recover the highly polar PIP3 species, you may need a proper derivatisation process like this methylation, afterwards the sensitivity is much better, in this paper PIP3 from tissue is described as well..http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460242/
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