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Reproducibility of Phosphoinositide Lipid Extraction & Presentation in Bioanalytical Assays
Author(s) -
Branch Angie M.,
Neilsen Paul O.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb180
Subject(s) - coefficient of variation , extraction (chemistry) , bioanalysis , phosphatidylinositol , chemistry , sample preparation , chromatography , reproducibility , biology , biochemistry , kinase
Phosphatidylinositol 4‐phosphate (PI4P) produced by Phosphatidylinositol 4‐Phosphate kinases is an abundant Golgi phosphoinositide functioning in lipid exchange and vesicle transport within mammalian cells. Researchers wanting to quantify PI4P use the Bligh and Dyer method for extracting and separating phospholipids in cells and biological tissues. This common lipid extraction procedure is known to have inherent variation. Using a human promyelocytic leukemia cell line and an ELISA that is sensitive and specific for PI4P, we examine sources of variation in bioassays including sample (HL‐60 cells), the extraction procedure, and lipid detection. We determined a PI4P Mass ELISA has low coefficient of variation (<10%) if the extracted sample volume is held constant and samples are prepped and lipids presented with standardized procedures. Thus, the organic solvent extraction process is the major source of variation in typical Bligh‐Dyer preparations. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .