Instantblue coomassie stain

SOD1 enzymatic activity was determined using in-gel zymography as previously described^{57 (link)}. Briefly, lumbar spinal cord PBS-soluble fractions (2.5 μg) and 100 ng of purified recombinant human SOD1^WT protein was separated in 8% native PAGE gels. Subsequently, gels were incubated in 5 mM nitrotetrazolium blue chloride (Sigma-Aldrich) for 20 min with gentle agitation, before incubation in developer solution (10 mM tetramethylethylenediamine and 30 µM riboflavin) for 15 min. Gels were developed by exposure to fluorescent light until sufficient contrast between the achromatic zones and background was achieved and images captured using a calibrated densitometer (GS-900; Bio-Rad, Australia). SOD1 activity was quantified as absorbance of the clear bands using ImageJ software (version 1.48, https://imagej.nih.gov/ij/). Each sample was run in duplicate. To account for equal protein between samples, SOD1 activity was normalised to InstantBlue Coomassie stain (Sigma-Aldrich) from samples run on an adjacent gel.

Purified samples were analysed by SDS-PAGE and western blot. Samples were applied to precast TGX gels (Bio-Rad, Hercules, CA, USA) before staining with InstantBlue Coomassie stain (Sigma-Aldrich) or immediately transferred to PVDF membrane (Bio-Rad) for western blot analysis. Western blots were stained with primary rabbit anti-GNAS (Novus Biologicals, Littleton, CO, USA NBP1-31730), primary mouse anti-His antibody (Qiagen, Hilden, Germany, 34660), secondary goat anti-rabbit 800CW antibody (LI-COR Biosciences, Lincoln, NE, USA, 926–32211), secondary anti-mouse 680CW antibody (LI-COR Biosciences, 926–68070) and an in-house made Alexa Fluor 488 conjugated mouse anti-flag antibody. Western blots were imaged on a Typhoon 5 imaging system (Amersham, Little Chalfont, UK).