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Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2557))

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Abstract

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.

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Acknowledgments

This work was supported by NIH grants GM070736 and GM141233 to H.F.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Margaret Kell, Abby Halpern & Heike Fölsch

Authors
  1. Margaret Kell
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  2. Abby Halpern
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  3. Heike Fölsch
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Corresponding author

Correspondence to Heike Fölsch .

Editor information

Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Yanzhuang Wang

  2. Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Vladimir V Lupashin

  3. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Todd R. Graham

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Kell, M., Halpern, A., Fölsch, H. (2023). Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_37

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  • DOI: https://doi.org/10.1007/978-1-0716-2639-9_37

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2638-2

  • Online ISBN: 978-1-0716-2639-9

  • eBook Packages: Springer Protocols

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