High-resolution clear native electrophoresis

HEK293T cells were transfected with 3–5 µg of plasmid DNA using the calcium phosphate method and treated as described previously (Guzman et al., 2017). In brief, cells were washed with ice-cold PBS and incubated for 15 min with a lysis buffer containing 0.1 M sodium phosphate, either 0.5% (w/v) digitonin or 20 mM DDM, protease inhibitors, and 20 mM iodoacetamide. The buffer was then transferred into a 1.5 ml Eppendorf tube. After centrifugation at 4 °C, an aliquot of the supernatant containing approximately 10 µg of protein was loaded into a native gel. The 4–14% acrylamide gradient gels were prepared as described (Wittig et al., 2007). The anode buffer contained 25 mM imidazole/HCl, pH 7.0, and the cathode buffer contained 50 mM tricine, 7.5 mM imidazole/HCl, pH 7.0 supplemented with the anionic detergent DOC (0.05%), and the non-ionic detergent Triton X-100 (0.05%) (Wittig et al., 2007). The gels were run in a cold room (4 °C) and the starting voltage was set to 100 V. After 1 hr, the voltage was increased to 150 V for another 2 hr. Gels were scanned on a fluorescence gel scanner (Typhoon FLA 9500, GE Healthcare, Freiburg, Germany) at 100 µm resolution. mVenus was excited with the built-in 473 nm laser and the emission was filtered with a 530/20 bandpass filter. Gel images were visualized using Fiji (Schindelin et al., 2012).