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Single-cell in situ imaging of palmitoylation in fatty-acylated proteins

Nature Protocols volume 9, pages 2607–2623 (2014)Cite this article

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Abstract

Dissecting the subcellular distribution of a fatty-acylated protein is key to the understanding of the molecular mechanisms regulating protein movement and function in a cell. This protocol describes how to perform single-cell imaging of palmitoylation in a fatty-acylated protein of interest with high sensitivity using click chemistry, proximity ligation and fluorescence microscopy. The initial steps in this protocol involve optimization of conditions for (i) metabolic incorporation of an alkynyl analog of palmitic acid into cellular proteins coupled with click chemistry and (ii) detecting a specific protein of interest with primary antibodies using automated fluorescence microscopy, followed by (iii) imaging palmitoylation of the target fatty-acylated protein of interest, such as Wnt, Sonic Hedgehog or H-Ras. Furthermore, we outline strategies for imaging specific fatty-acylated proteins with subcellular organelles and/or total proteome palmitoylation, and we discuss special considerations that need to be given depending on the experimental design. The use of clickable fatty acids with proximity ligation may have promising applications to the investigation of fatty acylation cell biology. The entire protocol takes ∼3 weeks to complete.

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Figure 1: Development of the click chemistry–proximity ligation imaging protocol.
Figure 2: General workflow of the assay.
Figure 3: Experimental design for the simultaneous detection of various relevant targets.
Figure 4: Experimental setup for sample treatment on coverslips using a humidity chamber.
Figure 5: Examples of the click chemistry–proximity ligation imaging method applied to the visualization of various targets.

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Acknowledgements

We acknowledge the use of microscopes at the Center for Advanced Light Microscopy (CALM) at Genentech.

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Authors and Affiliations

  1. Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA

    Xinxin Gao & Rami N Hannoush

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  1. Xinxin Gao
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Contributions

X.G. performed the experiments. X.G. and R.N.H. designed the study and wrote the paper.

Corresponding author

Correspondence to Rami N Hannoush.

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Gao, X., Hannoush, R. Single-cell in situ imaging of palmitoylation in fatty-acylated proteins. Nat Protoc 9, 2607–2623 (2014). https://doi.org/10.1038/nprot.2014.179

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