EMSA was used to verify the HiTS-RAP measured binding affinities of wild-type and mutant GFP binding aptamers to EGFP. To this end, in vitro transcribed aptamers were 3′ end-labeled with AlexaFluor647 Hydrazide (Invitrogen) as described elsewhere5 (link) and quantified by Qubit Fluorometer (Invitrogen). Fluorescently labeled aptamers were mixed with recombinant GST-EGFP protein at 25°C for 45 min. The GST-EGFP concentration in the binding reaction was varied as a 2/3 dilution series starting from 500 nM, and a no protein control. The final 20 μl binding reaction was composed of 1X PBS, 5 mM MgCl2, 0.4 U of Superase In, 1 μg of yeast tRNA, 0.005% NP-40, and 5 nM fluorescently-labeled aptamer. After addition of Bromocresol Green containing 30% glycerol to 6% final glycerol concentration, binding reactions were loaded on a 6% polyacrylamide gel (0.5X TBE, 5 mM MgCl2) that was pre-equilibrated to 4°C and pre-run at 120V for 10 min at 4°C. Loaded gels were run at 120V for 90 min at 4°C, and then imaged with a Typhoon 9400 scanner using Cy5 settings. Images were quantified by ImageQuant5.2 software, and data were fitted to Hill Equation to determine the Kd values using Igor software. EMSA was carried out with fluorescein labeled minimal NELFapt as described elsewhere17 (link).