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Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization   

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Abstract

Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads.

Keywords: Pulldown assay, Protein-protein interactions, Streptavidin bead, Biotinylated protein, Homooligomeric complex

Background

The amyloid precursor protein (APP) can form a homodimer through its large extracellular domain as well as its transmembrane domain, which plays an important role in biological function. The current protocol has been used in characterizing homo-dimerization of the APP transmembrane C-terminal 99 amino acid fragment (C99) (Yan et al., 2017). The basic principle of this assay is shown in Figure 1: The streptavidin-coated MagBeads can trap the biotinylated protein, which can pull down the interaction protein and detected by anti-FLAG antibody.


Figure 1. The principle of MagBeads-based pull-down assay. In this particular protocol, biotinylated Avi-tagged C99 proteins and associated C99-TEV site-rTA-Flag protein were used.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xu, T., Yan, Y., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization. Bio-protocol 7(22): e2901. DOI: 10.21769/BioProtoc.2901.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017a). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem, 292: 15826-15837.
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