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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Curr Protoc Cell Biol. 2018 Sep 25;81(1):e63. doi: 10.1002/cpcb.63

Figure 3.

Figure 3

A FRET assay to measure TA transfer from Sgt2 to Get3. (A) Scheme of the FRET assay to monitor TA loading onto Get3. Green and red stars denote the donor and acceptor dyes, respectively. (B) Sample SDS-PAGE of the Get3 labeling reaction stained with Coomassie blue. Lane 1 is Get3-ybbR labeled with BODIPY-FL-CoA via Sfp-mediated conjugation. Lane 2 is unlabeled Get3-ybbR. (C) Representative donor fluorescence emission spectra for the purified Sgt2•TACm complex (purple; donor only), Sgt2•TACm incubated with unlabeled Get3, Get4/5 and ATP (blue; donor + unlabeled acceptor), Get3BDP and Get4/5 (magenta; acceptor only), and Sgt2•TACm complex incubated with Get3BDP, Get4/5 and ATP (green; donor + acceptor). Adapted from Figure 6B in (Rao et al., 2016). (D) Representative data for equilibrium titrations of TA transfer from Sgt2 to Get3. Adapted from the data with the 2AG substrate in Figure 6E in (Rao et al., 2016).