Mops sds running buffer

Cells were manually lysed in N-Per Buffer (ThermoFisher Scientific #87792) with 1× Halt protease and phosphatase inhibitor (ThermoFisher Scientific #78442) for 10 min at room temperature, followed by a 14,000×g centrifugation at 4 °C to remove cellular debris. Total protein concentrations were measured with the Pierce BCA Assay, following the manufacturer’s instructions (ThermoFisher Scientific #23227). Twenty micrograms of protein were prepared with NuPAGE LDS sample buffer (ThermoFisher Scientific #NP0007) and NuPAGE reducing agent (ThermoFisher Scientific #NP0004). Samples were heated at 95 °C for 5 min, and loaded on 4–12% Bis–Tris gels for SDS-PAGE (ThermoFisher #NP0336BOX). Gels were run in 1× MOPS SDS running buffer (ThermoFisher Scientific #NP0001) and transferred to nitrocellulose using a wet transfer system XCellII Blot Module (ThermoFisher Scientific, #EI9051). Membranes were blocked in Odyssey blocking buffer (Licor #927-50000) for 1 h at room temperature. Membranes were incubated with primary antibodies for 16 h at 4 °C diluted in Odyssey blocking buffer. The following day, membranes were incubated 1 h at room temperature with the corresponding species-specific IRDye secondary antibodies diluted in Odyssey blocking buffer and imaged on Licor Odyssey CL.

Samples were loaded on NuPAGE Bis-Tris 4–12% gradient polyacrylamide gels (Thermo Fisher Scientific). SDS-PAGE was conducted in either MES or MOPS SDS running buffer (Thermo Fisher Scientific) at 200 V for 40 min. As a molecular weight marker, a pre-stained PageRuler was used (Thermo Fisher Scientific). Proteins were then transferred to a methanol-activated PVDF membrane (Millipore) for 1 hr at 100 V, in blot buffer (40 mM glycine, 25 mM Tris base, and 20% methanol). After protein transfer, PVDF membranes were blocked for 1 hr at room temperature with PBS containing 5% milk. After that, two washing steps of 5 min each were performed in PBS-T (PBS supplemented with 0.05% Tween-20) at room temperature prior to the addition of a primary antibody solution (in PBS-T supplemented with 2% BSA and 0.02% Na-azide). After 1 hr incubation at room temperature, blots were washed four times with PBS-T prior to the addition of fluorescently labeled secondary antibodies (goat anti-mouse AlexaFluor 680 [Thermo Fisher Scientific] and goat anti-rabbit IRDye 800CW [LI-COR Biosciences]). After 30 min incubation at room temperature, blots were washed four times with PBS-T and once with PBS. Imaging was performed with the LI-COR Odyssey CLx system and analyzed with Image Studio Lite (version 5.0.21, LI-COR Biosciences).

Proteins were separated using SDS-PAGE (NuPAGE® 4–12% Bis-Tris; Thermo Fischer Scientific) in MOPS SDS Running Buffer (NuPAGE®, Thermo Fischer Scientific) and transferred to nitrocellulose membrane using iBlot™ Gel Transfer Stacks (Thermo Fischer Scientific). Membranes were blocked in 5% skimmed milk buffer in TBST (0.05 Tris-base, 0.5 M NaCl with 0.1% Tween-20) and incubated with primary antibodies diluted in blocking buffer overnight at 4°C. On the next day, the membranes were washed three times with 0.5% skimmed milk in TBST, incubated with HRP-conjugated secondary antibodies for 1 h at room temperature (RT), and washed again 5 times. Proteins were visualized using ECL plus Western blotting detection system (GE Healthcare) with a Fuji film LAS3000 system. The following antibodies were used: Rabbit anti-LDLR (Abcam ab52818, 1:600), Mouse anti-beta actin (Sigma A5441, 1:5000). Secondary HRP-linked antibodies were used in 1:2000 dilution (Dako P0260, Cell signaling 7074S).

Cells from 1 ml culture were centrifuged and 1X LDS sample buffer with reducing agent (Thermo Fisher) was added to the cell pellet, sufficient to give an OD₆₀₀ of 2.5. Samples were boiled and resolved on a NuPAGE 10% Bis-Tris Gel in MOPS SDS Running Buffer from Thermo Fisher. Proteins were transferred onto a nitrocellulose membrane with an iBlot gel transfer system. Lysine-acetylated proteins were detected with mouse anti-AcK antibody (Cell Signaling Technology, cat. # 9681S) diluted 1/1000 in TBS-T with 5% of BSA, incubated overnight at 4°C and developed with peroxidase conjugated anti-mouse antibody. The signal was detected with a chemiluminescent reaction, using ECL^TM Western Blotting kit from GE Healthcare, and detected by a BioRad Molecular Imager ChemiDoc XRS System.