Nupage lds sample buffer 4x

Total cell lysates were prepared in RIPA Buffer (10 mM NaPO₄, pH 7.2, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Na-deoxycholate, 1% Nonidet P40) containing Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche). Lysates were solubilized in NuPAGE LDS Sample Buffer (4X) (Life Technologies) with NuPAGE Sample Reducing Agent (10X) (Life Technologies) added to 1X. Lysates were separated by electrophoresis with 20 µg of protein per lane in a NuPAGE 4–12% Bis-Tris Gel (Life Technologies) and transferred to an Immobilon-FL 0.45 µm Pore Size Transfer Membrane (Millipore) using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). Membranes were blocked and all antibody dilutions were performed in 5% BSA (Sigma) in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). All washes were performed in TBST. Primary antibodies included α-tubulin #05829 (Upstate Biotechnology), HER2 #MS-325 (Neomarkers), EGFR #4267 (Cell Signaling Technology), PAK1 #2602 (Cell Signaling Technology), Src #2110 (Cell Signaling Technology), Phospho-Src Y416 #6943 (Cell Signaling Technology), FAK #3285 (Cell Signaling Technology), and FAK Y576/577 #3281 (Cell Signaling Technology). Secondary antibodies were Alexa Fluor Conjugated Affinity Purified Anti-Rabbit or Anti-Mouse IgG (Life Technologies) detected using an Odyssey Infrared Imaging System (LiCor Biosciences).

10 kDa centrifuge filters were purchased from Sigma-Aldrich. Five ml HisTrap FF crude columns were bought from GE Healthcare. NuPAGE MES SDS Running Buffer (20 X), SeeBlue Plus2 Pre-stained Protein Standard, SimpleBlueTM SafeStain, NuPAGE Sample Reducing Agent (10 X), NuPAGE LDS Sample Buffer (4 X) and NuPAGENovex 4–12% Bis-Tris Protein Gels (1.0 mm 10 well) were obtained from Life Technologies. Quick StartTM Bradford 1 x Dye Reagent was from Bio-Raid. SnakeSkin Dialysis Tubing was purchased from Thermo Scientific. Linbro plates were bought from Hampton Research. NT. 115 premium capillaries were obtained from Nanotemper. NSP10 expression clones for mutants T12I, T102I, and A104V optimized for E. coli expression was purchased from Genscript.

C2C12 cell pellets in GM and after 24 h of DM were lysed in hypotonic buffer (20 mM HEPES pH 7, 0.15 mM EDTA, 0.15 mM EGTA, 10 mM KCl) with freshly added spermine, spermidine (0.15 mM, 0.5 mM, respectively) and protease inhibitor cocktail (Roche Applied Science). Cell lysates were centrifuged at 9000 rpm for 7 mn. The pellets were suspended in sucrose buffer (20 mM Tris pH 7.65; 60 mM NaCl; 15 mM KCl; 0.34 M Sucrose) and then in high salt buffer (20 mM Tris-HCl pH 7.65; 0.2 mM EDTA; 25% glycerol; 900 mM NaCl; 1.5 mM MgCl₂) to a final NaCl concentration of 300 mM. The nuclear extracts were treated with Micrococcal nuclease (0.0025 U/µl) at 37°C during 10 mn and sonicated during 10 mn at high frequency. The lysates were ultracentrifuged at 40000 rpm for 30 mn (Optima MAX-XP, Beckman Coulter) and pre-cleared during 1 h. Immunoprecipitations were carried out overnight at 4°C using 5 µg of each antibody. The second day, the immunocomplexes are washed four times in wash buffer (50 mM Tris-HCl, pH 7.65, 150 mM NaCl, Triton X-100 0.5%) and the proteins were eluted in NuPAGE LDS Sample Buffer (4X) (Life Technologies) at 96°C during 5 min. Finally, the immunoprecipitates were examined by western blot.

Fly tissues were resuspended in RIPA buffer, followed by grinding and centrifugation to remove debris. The supernatant was measured by Bradford assay, and 25–50 μg of protein in NuPAGE LDS sample buffer (4x) (NP0007, Life Technologies, Carlsbad, CA, USA) was loaded in each lane. NuPAGE Novex 4-12% Bis-Tris gel (#NP0321BOX, Life Technologies, Carlsbad, CA, USA) were loaded and run in 1xNuPAGE MES SDS Running Buffer (#NP0002, Life Technologies, Carlsbad, CA, USA). Western transfer was performed on PVDF membrane, followed by blocking in 5% milk/TBST for 1 h at 4 °C. The membrane was incubated with primary antibody (anti-Nbr, 1/2000) at 4 °C overnight. After washing the membrane in TBST buffer 3 times (5 min each), the membrane was incubated with secondary antibody (goat anti-rabbit IgG-HRP, #sc-2030, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4 °C for 2 h. The membrane was washed in TBST 3 times (5 min each), followed by signal development by Pierce ECL plus Western Blotting Substrate (#32132; Thermo Fisher Scientific, Waltham, MA, USA). The image was scanned by Fujifilm LAS-3000 Imager (Fujifilm, Tokyo, Japan).