Mes buffer system

Brains from wild-type and NSG1 KO mice were homogenized in 5mM CHAPS, 50mM Tris-HCl, 150mM NaCl, 1 × proteinase inhibitor with a Kontes microtube pellet pestle while kept on ice. Detergent-insoluble material was removed by centrifugation at 13,000 × g for 10 min. Aliquots of supernatants were mixed with SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.02 percent bromophenol blue, and 125 mM dithiothreitol) and incubated at 50°C for 20 minutes. The samples were separated on a 10% NuPage Bis-Tris gel (Invitrogen) using a MES buffer system (Invitrogen), transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad), blocked in Odyssey Blocking Buffer (LI-COR), and probed simultaneously with the primary antibodies: mouse anti-NSG1 (Santa Cruz; sc-390654, 1:1000) and rabbit anti-NSG2 (Abcam; ab189513, 1:1000) followed by incubation with the IRDye conjugated secondary antibodies: goat anti-mouse800CW (LI-COR; 926–32210, 1:10,000) and goat anti-rabbit680RD (LI-COR; 926–68071, 1:10,000). The Odyssey CLx infrared imaging equipment was used to detect infrared signals

25mg of aortic sample were lysed in 100μL of modified radioimmunoprecipitation buffer (2% SDS, 150mM NaCl, 50mM Tris pH8, 1X Roche Complete) and heating at 100°C for 1hr. The protein concentration of the cleared lysates was determined by Qubit fluorometry. 10μg of each sample was processed by SDS-PAGE using a 10% Bis-Tris NuPage Mini-gel with the MES buffer system (Invitrogen). The gel was run 5cm and each gel lane was excised into ten equally sized bands. Gel bands were processed by in-gel digestion with trypsin using a ProGest robot (DigiLab) with the following protocol: (a) Washed with 25mM ammonium bicarbonate followed by acetonitrile; (b) Reduced with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at RT; (c) Digested with sequencing grade trypsin (Promega) at 37°C for 4h; (d) Quenched with formic acid and analyzed without further processing.