Novex empty gel cassettes

In vivo MAVS aggregation assay was performed according to a published protocol (Hou et al., 2011 (link)). Briefly, crude mitochondria isolated using mitochondria isolation kit (89874, Thermo) were resuspended in 1x sample buffer (0.5 x TBE, 10% glycerol, 2% SDS) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate decahydrate, 1 mM β-glycerophophate) followed by protein concentration quantification using BCA method. Equal amounts of crude mitochondria were mixed with 1/10 volume gel loading dye (7025, NEB) and subjected to Semi-Denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE). Samples were loaded onto a homemade vertical 1.5% agarose gel prepared using Novex empty gel cassettes (NC2015, Thermo), empty gel cassette combs (NC3515, Thermo) and Bio-Rad agarose (1613101, BioRad). After electrophoresis in the running buffer (1 x TBE and 0.1% SDS) for 40 min with a constant voltage of 100 V at 4°C, the proteins were transferred to 0.2 μM Amersham Protran Premium Nitrocellulose Western Blotting Membranes (10600004, GE) at 400 mA for 3 h in ice cold transfer buffer (10% methanol, 191 mM glycine, 25 mM tris-base, 0.1% SDS, pH 8.3) followed by immunoblotting.