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Nat Protoc. 2016 Jun;11(6):1021-38. doi: 10.1038/nprot.2016.059. Epub 2016 May 5.

A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

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European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
European Molecular Biology Laboratory (EMBL) Cell Biology and Biophysics Unit, Heidelberg, Germany.
European Molecular Biology Laboratory (EMBL) Advanced Light Microscopy Facility, Heidelberg, Germany.


Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.

[Indexed for MEDLINE]

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