(a) In vivo protein expression (left panel) and solubility screens (right panel) for the sfCherry-GFP(10–11) hairpin inserted at Pro52/Gly53 and Asp169/Gly170. Pictures were taken of the plates after 4 h of co-induction (to monitor protein expression by GFP fluorescence) and after 2 h of induction with anhydrotetracycline (AnTet) followed by 1 h rest and 1 h induction of GFP(1–9) (to monitor soluble protein by GFP fluorescence). Fluorescence from folded sfCherry was monitored using 550 nm excitation/610 nm emission (red fluorescence) and reconstituted GFP fluorescence was monitored using 488 nm excitation/520 nm emission (green fluorescence). Pictures are shown with 0.5 s exposure times for red fluorescence and 0.25 s exposure times for green fluorescence. (b) In vitro sensitivity characterization of sfCherry-GFP(10–11) complementation with GFP(1–9). 20 µl aliquots containing 1.56–200 pmol of sfCherry-GFP(10–11) hairpin were mixed with 180 µl aliquots containing 800 pmol GFP(1–9) to start the complementation. AU, arbitrary fluorescence units. (c) Superimposition of scaled progress curves for complementation of 200, 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 pmol samples. The curves can be superimposed well by linear scaling, indicating that the shape of the progress curves does not depend on the concentration of the tagged protein or the depletion of the pool of unbound GFP(1–9) fragment (see §3).