Fig. 2.

Detection of specific protein-ligand interactions by DRaCALA. (A) DRaCALA images of interactions between purified proteins (20 μM) incubated with 500 nM ¹⁴C-cAMP, 4 nM [³²P]ATP, or 4 nM ³²P-cdiGMP. Protein-ligand mixtures were spotted on nitrocellulose and allowed to dry before imaging using a Fuji FLA7100 PhosphorImager. Cognate protein-nucleotide combinations are indicated by arrowheads. MBP was used as a negative control. (B) DRaCALA images of competition assays assessing the ability of 1 mM indicated cold nucleotides to compete with binding interactions between 4 nM ³²P-cdiGMP and 2.5 μM HisMBP-Alg44_PilZ. (C) Graph of F_B for each sample in Fig. 1B, with averages indicated by a horizontal bar. NC, no competitor. P values were determined by a Student t test for significant differences compared with the NC control for three independent experiments. The I_total of each DRaCALA spot in Fig. 2 A and B is provided in Tables S1 and S2, respectively.