Ecl imager genegnome xrq

Protein content in cell lysates was determined using a bicinchoninic acid (BCA) kit according to the manufacturer’s instructions (Thermo Fisher Pierce). Samples were solubilized in sample loading buffer (RotiLoad, Roth, Germany) at concentrations of approximately 1.5 μg/μl and run on 4–20% SDS-Polyacrylamide gels. For immunodetection proteins were transfered onto nitrocellulose and incubated with the primary antibodies as indicated. Immunoreactivity was detected according to standard protocols using an ECL Imager (GeneGnome XRQ, Syngene, Cambridge, UK) or ECL-films. Images below saturation or films with the shortest exposure time still showing all expected bands were used for quantification by Image-J. For each blot, band intensities were normalized relative to the respective loading control and statistically analyzed using Prism software.

Cell pellets were homogenized by incubation at 4 °C for 30 min in a Triton-homogenization buffer (i.e., 20 mM of Tris pH 7.5, 150 mM of NaCl, 1% Triton-X-100, 2 mM of MgCl₂, 750 U/mL of Benzonase (Sigma, Darmstadt, Germany), and protease inhibitors (cOmplete^TM Protease Inhibitor cocktail tablets, Roche, Mannheim, Germany), centrifuged at 15,000× g for 20 min, and the resulting supernatants were used for the analysis. The protein content in the supernatant was determined using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The samples were denatured (at 95 °C, for 5 min) in a sample loading buffer (RotiLoad I, Roth, Karlsruhe, Germany) at concentrations of approximately 0.8–1.5 µg/µL and run on SERVAGel^TM TG PRiME^TM 4–20% Gels (Serva, Heidelberg, Germany). The proteins were then immunoblotted according to the standard procedures and the immunoreactivity was detected using an ECL Imager (GeneGnome XRQ, Syngene, Cambridge, UK). The quantification of band intensities was performed using ImageJ, and the OD values were normalized to either ERK1/2 or β-actin as the loading controls.