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See & Catch Method for Studying Protein Complexes in Yeast Cells: A Technique Unifying Fluorescence Microscopy and Mass Spectrometry

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Yeast Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1163))

Abstract

We have developed a method for studying proteins and protein complexes in yeast cells based on unification of fluorescence microscopy and mass spectrometry techniques. To apply the method, termed by us as “See & Catch,” we first produced a variety of DNA plasmids used as PCR templates for genomic tagging of proteins with a modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. Among those modular tags, we found several combinations which were optimal for determining protein subcellular localization and for purifying the tagged proteins and protein complexes for detailed analysis by mass spectrometry. Combining fluorescence microscopy and mass spectrometry into a single method provides a unique possibility to obtain a unified view of the processes regulating dynamic properties of the proteins and protein complexes in living cells.

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Acknowledgement

We thank Roger Tsien’s laboratory for sharing DNA coding for several fluorescence proteins. This work was supported by the Sandler Family Fund.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 94143, USA

    Changhui Deng & Andrew N. Krutchinsky

  2. The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA

    Andrew N. Krutchinsky

Authors
  1. Changhui Deng
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  2. Andrew N. Krutchinsky
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Corresponding author

Correspondence to Andrew N. Krutchinsky .

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Editors and Affiliations

  1. Capital Normal University College of Life Sciences, Beijing, China

    Wei Xiao

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Deng, C., Krutchinsky, A.N. (2014). See & Catch Method for Studying Protein Complexes in Yeast Cells: A Technique Unifying Fluorescence Microscopy and Mass Spectrometry. In: Xiao, W. (eds) Yeast Protocols. Methods in Molecular Biology, vol 1163. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0799-1_6

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  • DOI: https://doi.org/10.1007/978-1-4939-0799-1_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0798-4

  • Online ISBN: 978-1-4939-0799-1

  • eBook Packages: Springer Protocols

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