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. Author manuscript; available in PMC: 2014 Sep 19.
Published in final edited form as: J Biomol Screen. 2013 Apr 15;18(8):910–920. doi: 10.1177/1087057113485789

Figure 1.

Figure 1

(A) Format of the AlphaScreen assay to detect Skp2-Cks1 interactions. Binding of the glutathione S-transferase (GST)– Skp2/Skp2 complex and His6-tagged Cks1 brings the beads together by immobilization to GST-coated donor beads and nickel-chelated acceptor beads, respectively. When excited at 680 nm, donor beads generate singlet oxygens, which diffuse to the acceptor beads, creating a chemiluminescent reaction that is measured as an AlphaScreen signal. The lower panel shows the format of the counterscreen AlphaScreen assay to eliminate false-positive hits. In this system, donor beads conjugated to a GST-biotin peptide served as a binding partner for streptavidin-conjugated acceptor beads. (B) A schematic of the SCFSkp2-dependent p27 ubiquitination pathway in vitro.