Anti c myc antibody clone 9e10

The binding assay was performed using Meso Scale Discovery 384 well high bind plates^{56 (link)}. All binding experiments were carried out at 22 °C. Liposomes (2 μL) were passively adsorbed on the electrode surface for 1 h, and residual sites on the surface were blocked for 1 h with 0.25% porcine gelatin (Sigma-Aldrich) in TRIS buffer (50 mM Tris, 150 mM NaCl, pH 8.0). After three washing steps with TRIS buffer, serial dilutions of recombinant protein in blocking buffer was added to the respective wells and incubated for 2 h. Unbound protein was removed, and anti-c-myc antibody (clone 9E10, Santa Cruz Biotechnology) at 1.25 µg/mL concentration in 0.25% porcine gelatin was applied for 1 h followed by three subsequent wash steps with TRIS buffer. For detection, a secondary anti-mouse antibodies labeled with Sulfo-TAG (Meso Scale Discovery) was used at 1.25 μg/mL in blocking buffer for 1 h in the dark. Free secondary antibody was washed off, and reading buffer (surfactant-free reading buffer from Meso Scale Discovery) was added. The readout was performed on a Meso Scale Discovery SECTOR Imager 6000 chemiluminescence reader. Data were analyzed with GraphPad Prism 6.07. First, signal from PC/cholesterol (70/30 mol %) vesicles was subtracted as a background. Next, a non-linear curve fitting was applied, and the binding kinetics were calculated using one site—specific binding fitting.

Human insulin (Humulin R) was obtained from Eli Lilly (Indianapolis, IN, U.S.A.). The analog of adenosine monophosphate 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was obtained from TOCRIS (catalogue #2840) and ionomycin was obtained from Sigma-Aldrich (catalogue #I9657). The primary monoclonal mouse anti-c-Myc antibody (clone 9E10) was purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.), and the polyclonal rabbit anti-c-Myc antibody was purchased from Millipore-Sigma (St. Louis, MO, U.S.A.). Secondary antibodies, including goat anti-rabbit IgG H&L (Alexa Fluor^® 647) and goat anti-rabbit IgG H&L (Cy3^®), were purchased from ABCAM, while goat anti-rabbit H&L HRP was from Thermo Scientific (Invitrogen^®). Sigma FAST OPD (o-phenylenediamine dihydrochloride peroxidase substrate) tablets were purchased from SIGMA-ALDRICH (Catalogue #P9187).