FIGURE 2.
Analysis of distinct protein–RNA complexes in one sample by NIR probes. (A) UV crosslinking of MS2-Atto680 (200 fmol) to MBP (3 μg) or indicated amounts of MS2BP after RNase A treatment and SDS-PAGE. Complex formation was analyzed by infrared scanning of the SDS-gel (A) followed by Western blotting with anti-MBP (B). (4×MS2BP) Tetramer, (2×MS2BP) dimer, (MS2BP) monomer, (MBP) maltose binding protein. (C) Competition of the MS2BP/MS2 association analyzed by EMSA. Binding of MS2-Atto680 (15 fmol) to MS2BP (80 nM) was competed by a 50-fold molar excess of unlabeled MS2 (lane 3) or MS2mut (lane 4). The percentage of bound probe (bottom panel) was determined as described in Figure 1. Sequence and stem–loop structure of used probes are indicated in Supplemental Figure 1A. (D) EMSA analysis of the MS2BP/MS2 and GST-λ/B-box association in one sample. Complex formation of MS2BP and/or GST-λ with MS2-DY776 (green, 25 fmol) and/or B-box-Atto680 (red, 15 fmol) was analyzed by EMSA. Note that both NIR RNAs were present in all samples. (Lane 1) Buffer control, (lane 2) MS2BP (80 nM), (lane 3) GST-λ (500 nM), (lane 4) MS2BP (80 nM) and GST-λ (500 nM). Free NIR probes and protein–RNA complexes are indicated. For color-separated scans, see Supplemental Figure 2C.