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. Author manuscript; available in PMC: 2011 Oct 21.
Published in final edited form as: J Am Chem Soc. 2011 Sep 8;133(39):15578–15592. doi: 10.1021/ja204650q

Figure 4.

Figure 4

Kd measurements of c-di-GMP by gel-shift and its various analogs using the competition gel-shift assay with radiolabeled c-di-GMP. (a) Representative gel-shift experiment for measuring the Kd of c-di-GMP for each class of RNA by direct binding. (b) c-di-GMP binding curves for the class I (G94(2AP) and class II riboswitches. (c) Representative competition gel-shift experiment. RNA, radiolabeled c-di-GMP and increasing concentrations of competitor analog are incubated until equilibrium is achieved. Free c-di-GMP is separated from RNA-bound c-di-GMP by native PAGE. (d) Sample binding curve from the competition gel-shift assay with c-GMP-IMP. Data is fit to an equation for competitive binding (equation 3) to determine the analog Kd.