Ez link tfpa peg3 biotin kit

GR, PR, or FLAG peptides were biotinylated using the EZ-Link TFPA-PEG3-Biotin kit (Thermo Fisher). Splicing reaction mixtures containing these peptides (10 µM) were separated on Sephacryl-S500 gel filtration columns in a buffer containing 20 mM Tris, pH 7.8, 60 mM KCl, 0.1% Triton X100, and 0.2 mM PMSF. Proteins in the gel filtration fractions were analyzed on NuPAGE Novex 4–12% Bis-Tris Gels (Thermo Fisher) followed by western blotting. Total RNAs from the fractions were analyzed on an 8% denaturing polyacrylamide gel stained with ethidium bromide. The data for rows FLAG, SNRPC, SNRPB2, SF3A1, SF3A2/3, FUS and TARDBP shown in Figure 2A and the RNA gel shown in Figure 2B were from the gel filtration column containing the GR peptide. Similar results were obtained with gel filtration fractions containing PR and FLAG peptides. Pulldowns were carried out from fractions 33–69 in the gel filtration buffer using streptavidin magnetic particles (Roche) at 4°C for 2 hours and washed 5 times with 1X PBS/0.1% Triton X100. Proteins associated with biotinylated peptides were labeled using tandem mass tag (TMT) (McAlister et al., 2012 (link)) and analyzed with an Orbitrap Fusion mass spectrometer coupled to a Proxeon EASY-nLC 1000 liquid chromatography (LC) pump (Thermo Fisher Scientific).