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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.

Author information

1
European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
2
European Molecular Biology Laboratory (EMBL) Cell Biology and Biophysics Unit, Heidelberg, Germany.
3
European Molecular Biology Laboratory (EMBL) Advanced Light Microscopy Facility, Heidelberg, Germany.

Abstract

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.

PMID:
27149326
DOI:
10.1038/nprot.2016.059
[Indexed for MEDLINE]

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