Skip to main content
. 2020 Mar 16;22(4):179–191. doi: 10.1016/j.neo.2020.02.003

Fig. 1.

Fig. 1

AlphaScreen based SAG-CUL5 neddylation assay. (A) Reaction schemes for AlphaScreen based SAG/CUL5 neddylation assay (see M&M for details). (B) Components omission test: The reaction mixture was placed in a 384-well plate, followed by addition of AlphaScreen Donor and Acceptor beads, and subjected to analysis with a Perkin-Elmer Envision reader. Only the complete reaction yielded high AlphaScreen signal (AlphaSignal). (C) MLN4924 titration: MLN4924 at various concentrations was added into the reaction mixture which generated a dose–response inhibition of AlphaSignal. (D) MLN4924 inhibition of in vitro CUL5 neddylation reaction: MLN4924 at various concentrations was added into the reaction mixture and subjected to PAGE and Coomassie-blue staining. (E-F) Heatmap and statistics for a High-throughput format assay: The AlphaScreen assay was carried out using automatic dispensers with CV <10% in Complete and No ATP groups, Signal/Noise ratio = 42.12, and Z′ factor = 0.87.