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.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Anti-FLAG Western blot analysis reveals the presence of C99-TEV site-FLAG in the input lysate and in the Streptavidin Bead pulldown material (Figure 2), suggesting C99 homooligomerization. A practical example for Streptavidin Bead Pulldown Assay was previously shown in Yan et al., 2017. Figure 2. Pull down assay validation of C99 oligomerization. Biotinylated Avi-tagged C99 proteins and associated C99(WT)-TEV site-rTA-Flag were recovered on Streptavidin MagBeads (GenScript) and eluted with SDS sample buffer, while the dimer interface mutants (T43P/V45P: ctrl1 and I45P/V46P: ctrl2) (Yan et al., 2017) were not. Co-purified C99-TEV site-rTA-Flag proteins were detected by anti-FLAG immunoblotting. β-actin input levels serve as loading controls.
Recipes
Acknowledgments
This work was supported by the Van Andel Research Institute, the National Natural Science Foundation of China (31300607, 31300245 and 91217311), Ministry of Science and Technology grants 2012ZX09301001, 2012CB910403, and 2013CB910600, XDB08020303, 2013ZX09507001, Shanghai Science and Technology Committee (13ZR1447600), Shanghai Rising-Star Program (14QA1404300), and the National Institute of Health grants DK071662 (H.E.X.), GM102545 and GM104212 (K. M.). The authors declare no conflict of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.