Nupage lds electrophoresis sample buffer

For Western blot analysis, cultured cells were washed in ice-cold PBS solution and prepared under reducing (50 mM DTT) conditions by using NuPAGE LDS electrophoresis sample buffer (Invitrogen-Life Technologies). Samples were separated on a 4–12% polyacrylamide gel (Invitrogen-Life Technologies) using MOPS-SDS running buffer. Proteins were electroblotted to nitrocellulose membranes and unspecific binding sites were blocked in TBS-T [10 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6] containing 5% (w/v) non-fat milk powder. The membranes were then probed overnight with a polyclonal rabbit anti-NDRG2 (Atlas Antibodies, HPA002896, 1:500) (4°C). Membranes were washed three times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark), and the signal was detected by chemiluminescence (Pierce ECL, Thermo Scientific, Rockford, IL). Equal protein loading was monitored by probing with a ß-actin specific antibody (Sigma-Aldrich (A5316), 1:2000).

Cell lysates were prepared in RIPA buffer and containing the Complete™ mixture of proteinase inhibitors (Roche) and Phosphatase inhibitor cocktail 2 (#P5726, Sigma-Aldrich). Equal amounts of total cellular proteins (10–50 µg/lane) or exosome proteins (~2 µg/lane) determined by the DC protein assay (Bio-Rad, Düsseldorf, Germany) were mixed with NuPAGE™LDS electrophoresis sample buffer (Invitrogen, ThermoFisher Scientific) supplemented with DTT as a reducing agent or left untreated. Denaturation of protein samples, electroblotting and Western blot analysis were essentially applied as described previously [42 (link)]. After blotting, the transfer of proteins to the membrane was shown in Ponceau S stain and unspecific binding sites were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline with Tween 20 (TBST). For detection of individual proteins, the primary antibodies were diluted in 2.5% (w/v) non-fat milk powder in TBST. All antibodies used in this study are listed in Table 1 and their specificity for proteins of mouse, rat and human origin tested (Supplementary Figure S1). Primary antibodies were visualized with anti-mouse, anti-rabbit or anti-goat IgG secondary antibodies (all from Santa Cruz Biotech, Santa Cruz, CA, USA) with the SuperSignal chemiluminescent substrate (Pierce, Bonn, Germany).

Total protein from human or mouse liver tissue, that had been snap-frozen in liquid nitrogen, was prepared as described before [8 (link)]. Equal amounts of total protein (100 μg/lane) or serum (5 μL for mice and 1 μL for human) were mixed with NuPAGE™ LDS electrophoresis sample buffer (Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany) implemented with dithiothreitol (DTT) as a reducing agent. Denaturation of protein samples, electroblotting and Western blot were applied as described previously [8 (link)]. The primary antibodies were diluted in 2.5% (w/v) non-fat milk powder in Tris-buffered saline with Tween 20 (TBST) prior of membrane incubation. The primary antibodies used in this study (Table 1) were visualized with anti-mouse, anti-rabbit or anti-goat IgG secondary antibodies (all from Santa Cruz Biotech, Santa Cruz, CA, USA) with the SuperSignal chemiluminescent substrate (Pierce, Bonn, Germany). α₂M was used as a loading control for serum samples as a relatively stable plasma protein [70 (link)]. Western blot results were densitometrically analysed using the publicly available ImageJ software (version 1.52a, https://imagej.nih.gov/ij/download.html).