FIGURE 5.

Large-scale preparation of protein–RNA cross-links. A total of 600 μL of complex H or complex A reaction mixtures containing 37 nM [APB⁺⁹B]-pre-mRNA-S were loaded on a 14-mL TST41.14 rotor. A total of 5 (H) or 8 (A) such gradients were taken, and the peaks were pooled for cross-linking and isolation of the product. (A,B) Comparison of input cross-links with eluate. For cross-linking, gradient fractions 7–10 (H) and 11–14 (A) were pooled, cross-linked, and digested with RNase T1. A 10-μL aliquot was withdrawn before the biotin–streptavidin selection, and to compare it with the eluted protein–RNA cross-links from the beads, a PhosphorImager picture was taken from the silver-stained gel in C. (Note: The gel in A ran for a longer time, so no degraded RNA is visible at the bottom.) (C) Silver-stained gel of material eluted from the beads. The 150-μL protein–RNA cross-links were fractionated on a 10% SDS–polyacrylamide gel. (M) A total of 2 μL (0.36 μg) of unstained molecular weight marker (Bio-Rad); the molecular weight is indicated at right. (1) FUSE 2; (2) hnRNP A2/B1.