Bis tris native page

For RNA binding assays, dsRNA (1 ng/μl) was incubated with protein (at indicated concentration) in buffer A at RT for 10 min, and the complex was analyzed on Bis-Tris native PAGE (Life Technologies) after staining with SybrGold stain (Life Technologies). For RIG-I binding assays, fluorescein-labeled RIG-I was first incubated with dsRNA (1 ng/μl) and subsequently with RIPLET (at indicated concentration) prior to analysis by Bis-Tris native PAGE (Life Technologies). SybrGold or fluorescein fluorescence was recorded using the scanner FLA9000 (Fuji) and analyzed with Multigauge (GE Healthcare).

For experiments requiring dsRNA, 112 bp dsRNA was used unless mentioned otherwise. For detection purpose, either Cy5-labeled dsRNA or fluorescein-labeled protein was used (as mentioned in the relevant experiments). For experiments where Cy5-labeled dsRNA was used, MDA5 (or other RLHs) and/or their variants at 0.25 μM were first incubated with dsRNA (1 ng/μl) for 30 min in 20 mM HEPES pH 7.5, 100 mM NaCl, 1.5 mM MgCl₂ and 2 mM DTT at RT with or without 2 mM ATP. This was followed by the addition of the TRIM proteins (TRIM65, RIPLET, TRIM14, TRIM16, TRIM25 or TRIM47) and/or their variants (as mentioned in the experiment). The mixture was further incubated at RT for 10 min and the samples were then analyzed on Bis-Tris native PAGE (Life Technologies). For experiments where fluorescin-labeled TRIM65 CC-PSpry was used, GST-tagged MDA5 variants (1.6 μM) were directly incubated with fluorescin-labeled TRIM65 CC-PSpry (0.8 μM) for 15 min at RT before analyzing on native PAGE. For experiments where fluorescin-labeled MDA5 was used, fluorescin-labeled MDA5 or its variants (0.6 μM) was incubated with TRIM65 (0.3 μM) in the presence or absence of 112 bp dsRNA (8 ng/μl) for 30 min, and the mixture was analyzed on native PAGE. The gels were imaged using either Cy5 or fluorescin fluorescence in iBright FL1000 (Invitrogen).