Abstract
BackgroundThe lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps.ResultsWith the extension package “flippant” to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results.Conclusions“flippant” allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.
Highlights
The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay
Input data and data processing To analyze raw data stemming from dithionite scramblase assays, “flippant” requires for each data point information on the path to the fluorimeter-generated spectrum, the amount of protein reconstituted into proteoliposomes, the assay volume before and after addition of dithionite, the amount of lipids present etc
Retinitis pigmentosa is a degenerative disease of the retina, the majority of cases of which is linked to mutant forms of the G-protein coupled receptor rhodopsin [20]
Summary
The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. Starting with the identification of baseline and postreduction fluorescence from fluorimeter-generated spectra, via the calculation of the underlying statistics and through publication-grade representation of the results, data analysis in the context of this assay has until now involved an extensive series of manual steps, at most supported by spreadsheet calculation facilities [8, 9, 14].
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