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. Author manuscript; available in PMC: 2009 Apr 15.
Published in final edited form as: Anal Biochem. 2007 Nov 28;375(2):364–366. doi: 10.1016/j.ab.2007.11.024

Figure 2. Detection of the interaction between the SUMO binding motif (SIM) and SUMO.

Figure 2

(A) Scheme of the AlphaScreen protein-protein interaction assay where the interaction between biotinylated-SIM and His-tagged SUMO is detected following the addition of streptavidin Donor beads and GSH Acceptor beads.

(B–C) Assay development. All regents were diluted in a buffer containing 25 mM Hepes pH 7.4, 100 mM NaCl, and 0.1 % Tween-20. To each well, 5 µL of GST-SUMO-1 or GSTSUMO-3 was added followed by 5 µL of biotinylated-SIM. The two binding partners were incubated together for one hour at room temperature, and then 5 µL of GSH Acceptor beads and 10 µL of streptavidin coated Donor beads were added to the wells. Plates were sealed and incubated in the dark at room temperature for an additional hour before reading.