Fig. 5: Analysis of folding reporters translated in Strep-Tactin-resin-assisted Ec CFS.

a Immunoblot of protein bands derived from unfractionated translation reactions (1 Total) or insoluble fraction from flow-through (2 FT-ins) probed with anti-GFP antibodies. Panel 3 shows Coomassie-stained resin-bound protein fractions (3 Bound), eluted and resolved on SDS-PAGE. Co- and post- denote the respective reaction formats, co-(+btn) contains 10 mM biotin to prevent protein capture onto resin. The band densities were obtained as non-saturated integrations of the respective image bands using ImageJ software and normalized either to “Total, co-“ for panels 1, 2 or to “Bound, co-“ for panel 3. b Fluorescence analysis of resin-bound (RES) and free-unbound (FT) fractions following co- and post-translational protein capture. Gray defines soluble fractions either eluted with biotin from the resin or remaining in the flow-through after centrifugation at 20 kg for 30 min at 4 °C. Black defines insoluble fractions remaining on resin or removed from flow-through following elution or centrifugation, respectively. Fluorescence of insoluble fraction does not account for misfolded protein and only provides the relative estimation of the degree of protein aggregation. Fluorescence units were converted to picomoles of GFP using the respective calibration curves (Supplementary Fig. 16A, C) and further adjusted to 1 ml of translation reaction (for FT) or to resin amount used for 1 ml of translation reaction (for RES). Results are plotted as means ± s.d. of n = 3 independent experiments. c DHFR activity analysis in eluted protein fractions following co- or post-translational capture before (co-/post-) or after (NB/Gdn) on-resin treatment of co-translationally captured protein with 1.6 M guanidine hydrochloride-containing buffer (Gdn) or with neutral buffer lacking denaturant (NB) for 2 h at room temperature. Initial rates of fluorescence change for NADPH-oxidation reactions were converted to picomoles of active DHFR using the respective calibration plot (Supplementary Fig. 16B) and further adjusted as in (b). The results are plotted as means ± s.d. of n = 3 independent experiments. d Fluorescence analysis of GFP-hDHFR fractions eluted with standard elution buffer (-tw) or with the buffer containing 0.125% Tween 20 (+tw). The results are represented as means ± s.d. of n = 3 independent experiments.