(A) Comparison of FCS (i.e. Cy5 quenching in FCS apparatus) and MST methods for measurement of binding of Δp6 Gag to dimeric Ψ 200 RNA. The Cy5-tagged RNA was dimerized as described (Comas-Garcia et al., 2017) and diluted into binding buffer B to a concentration of 7 nM. This buffer was composed of 50 mM phosphate, pH 7.0, 0.05% Tween 20, 0.1 mM PMSF, and 1 mM β-mercaptoethanol, together with either 0.15 M or 0.45 M NaCl. The sample was then divided and, after 16 hr at 4°C, used for binding measurements by FCS or MST. Both methods give very similar KDs, although the MST curves suggest somewhat higher cooperativity in the binding than FCS. (B) Binding of Δ16–99 Gag protein to the three RNAs used for the Virus-like-particle (VLP) assembly experiments. Ψ 150 RNA, MBSM second generation RNA, and Reverse Complement RNA were all treated as described (Comas-Garcia et al., 2017) for Ψ dimerization. They were then diluted into Assembly Buffer (20 mM Tris pH 7.5, 0.15M NaCl, 5 mM MgCl2, 1 µM ZnCl2, 0.1% Tween 20, 0.1 mM PMSF, and 1 mM DTT). Binding of Δ16–99 Gag to the RNAs was then measured by MST. The FCS data in Figure 2A was treated as in Figure 1B–D. All MST data results are the means of three independent experiments. Each data-point in each MST experiment is the mean of triplicate measurements.