Nupage novex bis tris pre cast gels

Western immunoblot analyses were done by standard techniques using NuPAGE Novex Bis-Tris pre-cast gels (Life Technologies, Carlsbad, CA). Tissues were homogenized in ice-cold buffer composed of 0.1 M KH₂PO₄, 2 mM EDTA, 2% Triton X-100 and protease inhibitor cocktail (Roche, Basel, Switzerland). Homogenates were centrifuged at 12 000 × g at 4o C and protein concentrations in supernatants were determined with the BCA assay (BioRad, Hercules, CA). After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA overnight. Protein levels of ETC. complexes were detected with a cocktail of mouse monoclonal antibodies specific to selected subunits of the complexes (Life Technologies). Custom-made antibodies specific to mitochondrial malate dehydrogenase (mMDH) were used for the loading control. Secondary IRDye antibodies were used for imaging (Licor Biosciences, Lincoln, NE). Membranes were scanned with an Odyssey CLx scanner. Band intensity analysis and data quantification were done with Image Studio software.

Samples were resolved using NuPAGE Novex Bis-Tris pre-cast gels (Life Technologies, 12 % 10-well NP0341BOX) using the BLUeye Pre-Stained Protein Ladder (Geneflow Ltd, S6-0024) as a protein size reference. Proteins were transferred onto Immobolin PVDF membranes (Millipore, IPFL00010) and protein gel blot analysis was performed using indicated antibodies and visualized on Hyperfilm ECL (GE Healthcare, 28-9068) using the ECL western blotting analysis system (GE Healthcare, RPN2109). Adobe Photoshop software was used to crop full blots and band integrated densities were quantified using the ImageJ software.

Immunoblotting was performed according to standard protocols (Life Technologies). Electrophoresis was run in 4–12% gradient NuPAGE Novex Bis-Tris Pre-Cast Gels (Life Technologies) and followed by protein transfer to a PDVF or nitrocellulose membrane (Life Technologies) according to standard protocols. Blocking was performed in PBS 1×, 0.2% Tween, 5% dry milk powder (Marvel). Membranes were stained with the primary and secondary antibodies diluted in the same blocking solution. Primary antibodies used were as follows: mouse monoclonal anti-β-actin, 1:20,000 (Abcam), rabbit anti-EphB1, 1:500 (Santa Cruz), rabbit anti-ephrin-B1, 1:1000 (Santa Cruz), mouse monoclonal anti-GFAP, 1:500 (Sigma-Aldrich), rabbit monoclonal anti-STAT3, 1:1000 (Cell Signalling), rabbit monoclonal anti-pSTAT3 (Tyr705), 1:1000 (Cell Signalling), rabbit anti-Trim30, 1:1000, (Novus). Detection was performed by exposing the membrane to the Amersham ECL Prime WB Detection Reagents (GE Healthcare).

Western blot was performed as previously described [22 (link)]. Briefly, either 25 mg of liver tissue or one-half of the cerebellum were lysed in 1 mL of buffer containing 1% SDS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates were centrifuged for 10 min at 12,000× g and 4 °C, and the supernatants were collected. Protein concentration was measured using a protein assay kit (see above). Lysates were then mixed with 1× lithium dodecyl sulfate (LDS) buffer and 1× reducing agent (Life Technologies) and heated for 10 min at 90 °C. Thirty micrograms of total cell lysate were separated by gel electrophoresis (NUPAGE Novex Bis-Tris precast gels, Life Technologies), using gels of 10%, 12%, or 4–12%, in an MOPS running buffer. Proteins were blotted on 0.2 μm nitrocellulose membranes (AmershamTM HybondTM-ECL, GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA). After incubation with the indicated primary and secondary antibodies, immunocomplexes were detected by chemiluminescence (PIERCE ECL, Thermo Scientific, Waltham, MA, USA). Densitometric measurements of the bands in the western blot analysis were evaluated using appropriate software (ImageJ 1.46r, National Institutes of Health, Bethesda, MD, USA), original western blots can be found at Figure S2.

Samples were prepared as indicated in the respective experimental sections and were resolved using NuPAGE Novex Tris-Acetate pre-cast gels (Life Technologies, 3–8% 10-well EA0375BOX, 3–8% 12-well EA03752BOX) or NuPAGE Novex Bis-Tris pre-cast gels (Life Technologies, 12% 10-well NP0341BOX, 12% 12-well NP0342BOX, 4–12% 10-well NP0321BOX, 12-well NP0321BOX). The BLUeye Pre-Stained Protein Ladder (Geneflow Ltd, S6-0024) was run alongside the samples for protein size reference. Proteins were transferred from gels onto Immobolin PVDF membranes (Millipore, IPFL00010). Western blot analysis was performed using antibodies listed in Supplementary Table 2 as outlined therein and visualized on Hyperfilm ECL (GE Healthcare, 28–9068) using the ECL western blotting analysis system (GE Healthcare, RPN2109). Adobe Photoshop software was used to crop full blots (Supplementary Figs 8 and 9). Band intensities were quantified using the ImageJ Gel analysis tool.