Native agarose gel electrophoresis (NAGE)

NAGE was performed in 1% agarose gels in 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) as previously described [9, 23, 99]. For routine detection of nucleic acids gels contained 0.5 μg/ml ethidium bromide (EB); subsequent protein staining was done using Coomassie Brilliant Blue R250 (CB) followed by extensive destaining in fixing buffer (50% MeOH, 7% acetic acid, v/v in H₂O).

Alternatively, gels run without EB were stained for RNA using 1x Sybr Green 2 (SG2) RNA stain in TAE buffer (from 10,000x in DMSO; FMC Bioproducts), followed by protein staining with ready-to-use Sypro Ruby (SR) Protein Gel Stain (BioRad). Fluorescence signals were recorded using a laser Scanner (Typhoon FLA 7000; GE Healthcare) set at excitation 473 nm / filter Y520 nm (SG2) and 473 nm / filter O580 nm (SR). Signals were quantified using ImageQuant software. Higher quantitation accuracy was achieved by several adapations of the protocol, as described next.