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Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in Plants

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Protein-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2690))

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Abstract

Protein complex immunoprecipitation (co-IP) is an in vitro technique used to study protein–protein interaction between two or more proteins. This method relies on affinity purification of recombinant epitope-tagged proteins followed by western blotting detection using tag-specific antibodies for the confirmation of positive interaction. The traditional co-IP method relies on the use of porous beaded support with immobilized antibodies to precipitate protein complexes. However, this method is time-consuming, labor-intensive, and provides lower reproducibility and yield of protein complexes. Here, we describe the implementation of magnetic beads and high-affinity anti-green fluorescent protein (GFP) antibodies to develop an in vitro GFP-protein trap-like system. This highly reproducible system utilizes a combination of small sample size, versatile lysis buffer, and lower amounts of magnetic beads to obtain protein complexes and aggregates that are compatible with functional assays, Western blotting, and mass spectrometry. In addition to protein–protein interactions, this versatile method can be employed to study protein–nucleic acid interactions. This protocol also highlights troubleshooting and includes recommendations to optimize its application.

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Acknowledgments

This research was funded by the National Science Foundation (IOS-2038872).

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Authors and Affiliations

  1. Department of Biology, University of Alabama, Birmingham, AL, USA

    Danish Diwan & Karolina M. Pajerowska-Mukhtar

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  1. Danish Diwan
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  2. Karolina M. Pajerowska-Mukhtar
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Corresponding author

Correspondence to Karolina M. Pajerowska-Mukhtar .

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Editors and Affiliations

  1. Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA

    Shahid Mukhtar

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Diwan, D., Pajerowska-Mukhtar, K.M. (2023). Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in Plants. In: Mukhtar, S. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 2690. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3327-4_5

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  • DOI: https://doi.org/10.1007/978-1-0716-3327-4_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3326-7

  • Online ISBN: 978-1-0716-3327-4

  • eBook Packages: Springer Protocols

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