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Determining the number of specific proteins in cellular compartments by quantitative microscopy

Nature Protocols volume 6, pages 1953–1968 (2011)Cite this article

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Abstract

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.

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Figure 1: Flowchart outlining the analysis of isolated cellular compartments.
Figure 2: Antibody labeling of proteins of interest in cellular compartments.
Figure 3: Imaging isolated cellular compartments.
Figure 4: Deconvolution/fitting of fully labeled sample using single-antibody distribution to obtain protein copy number.

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Acknowledgements

We gratefully acknowledge the support provided by the National Institutes of Health (NS052637 to D.T.C. and S.M.B.).

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Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, Washington, USA

    Sarah A Mutch, Jennifer C Gadd, Bryant S Fujimoto, Perry G Schiro & Daniel T Chiu

  2. Department of Pharmacology, University of Washington, Seattle, Washington, USA

    Patricia Kensel-Hammes & Sandra M Bajjalieh

Authors
  1. Sarah A Mutch
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  2. Jennifer C Gadd
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  3. Bryant S Fujimoto
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  4. Patricia Kensel-Hammes
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  5. Perry G Schiro
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  6. Sandra M Bajjalieh
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  7. Daniel T Chiu
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Contributions

S.A.M. designed and performed the experiments, worked on image and data analysis, and wrote the manuscript; J.C.G. performed all aspects of the experiments and helped to prepare the manuscript; B.S.F. wrote the image processing and statistical fitting programs and helped with data analysis; P.K.H. prepared the samples used in the experiment; P.G.S. worked on the design and construction of the microscopy setup and helped with image acquisition; S.M.B. and D.T.C provided overall input into the project and helped to prepare the manuscript.

Corresponding authors

Correspondence to Sandra M Bajjalieh or Daniel T Chiu.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Discussion 1

Discussion on semi-automated identification of regions of interest in images. (DOCX 10 kb)

Supplementary Discussion 2

Discussion on the fitting procedure. (DOCX 77 kb)

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Mutch, S., Gadd, J., Fujimoto, B. et al. Determining the number of specific proteins in cellular compartments by quantitative microscopy. Nat Protoc 6, 1953–1968 (2011). https://doi.org/10.1038/nprot.2011.414

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