Native PAGE conditions were prepared by casting 10 × 10 cm 10% PAGE gels (29:1 acrylamide:bisacrylamide; Sigma) using using native PAGE buffer, i.e. the same final buffer composition as used for all the in vitro assays (40 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2). After polymerization, the samples were run at 40 V for at least 2 h in a water-cooled PAGE chamber to keep the buffer temperature at ~25°C.
For staining, gels were incubated in buffer containing 10 μM DFHO for 15 min. Gels were then imaged to detect DFHO-Corn complexes. DFHO-stained gels were analyzed on a ChemiDoc MP imaging station (BioRad) at 470/30 nm excitation and 530/28 nm emission (green channel) and 530/28 nm excitation and 605/50 nm emission (red channel). Next, counterstaining with 1X SYBR Gold was performed for 15 min followed by gel imaging to detect all RNA species in each lane. SYBR Gold-stained bands were imaged using UV excitation (302 nm) and 590/110 nm emission on the ChemiDoc MP imaging station.
For staining, gels were incubated in buffer containing 10 μM DFHO for 15 min. Gels were then imaged to detect DFHO-Corn complexes. DFHO-stained gels were analyzed on a ChemiDoc MP imaging station (BioRad) at 470/30 nm excitation and 530/28 nm emission (green channel) and 530/28 nm excitation and 605/50 nm emission (red channel). Next, counterstaining with 1X SYBR Gold was performed for 15 min followed by gel imaging to detect all RNA species in each lane. SYBR Gold-stained bands were imaged using UV excitation (302 nm) and 590/110 nm emission on the ChemiDoc MP imaging station.