Nupage bis tris sds page

Tissue or cell culture samples were prepared in ice-cold Lysis Buffer (20 mM Tris, pH 7.4, 180 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X–100, 2.5 mM pyrophosphate, 50mM NaF, 5mM glyerco-phosphoate, 50nM calyculin A, 1mM Na₃VO₄ and supplemented with Complete EDTA–free protease inhibitor tablet (Roche). Lysates were sonicated, then were centrifuged at 16,000 g for 15 min at 4°C, and the supernatants were used for subsequent analyses. Protein concentration was determined using the bicinchoninic acid assay (Pierce). Protein lysates were denatured in Laemmli buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue, 0.7 M β–mercaptoethanol), resolved by 4–12% NuPAGE Bis-Tris SDS–PAGE (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF 0.45 m) membrane. Primary antibodies were diluted in TBS containing 0.05% Tween (TBS–T), 5% BSA, and 0.02% NaN₃. Membranes were incubated overnight with primary antibodies at 4°C. For secondary antibody incubation, anti-rabbit or anti-mouse HRP (Promega) was diluted in TBS–T containing 5% dry nonfat milk. Results were visualized with enhanced chemiluminescence (ECL) western blotting substrates (Pierce). Densitometry was performed using ImageJ 1.52k. For each image, signal was normalized such that the average of all control samples equaled 1.0.

Protein concentration was determined using the bicinchoninic acid assay (Pierce). 7.5 μg of protein sample was loaded in each lane for western blot analysis if not otherwise indicated. Protein samples were denatured in 1X NuPAGE LDS Sample Buffer (Invitrogen) without boiling, resolved by 4-12% NuPAGE BisTris SDS–PAGE (Invitrogen), and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibody incubations were performed in TBS containing 0.05% Tween (TBST) and 5% milk, for either 4 hr at room temperature or overnight at 4°C; secondary antibody incubations were performed using either horseradish peroxidase (HRP)-conjugated or IRDye secondary antibodies in TBST with 5% milk for 2 hr at room temperature, for visualization using Immobilon Crescendo Western HRP substrate (Millipore) or fluorescence signal respectively. A complete list of antibodies can be found below.

Protein lysates were prepared in MPER (ThermoScientific, Waltham, MA, USA) containing protease (Roche, Indianapolis, IN, USA) and phosphatase (Sigma-Aldrich, St. Louis, MO, USA) inhibitors. Lysates were resolved by 4-12% NuPage Bis-Tris SDS-PAGE (Invitrogen) and transferred to nitrocellulose. Antibodies: Rabbit anti-p44/42 MAPK (Erk1/2) (1:1,000, #4695 Cell Signaling Technology, Danvers, MA, USA), Rabbit anti-Phospho-p44/42 MAPK (Erk1/2), Thr202/Tyr204 (1:2,000, #4370, Cell Signaling Technology), Mouse anti-β-actin (1:3,000, #926-42212, LI-COR, Lincoln, NE, USA), IRDye 680RD Goat anti-Rabbit (1:15,000, 925-68071, LI-COR) and IRDye 800CW Donkey anti-Mouse (1:15,000, 92532212, LI-COR). Blots were developed using the LI-COR Odyssey CLx Imaging System.