Chemidoc xrs embellish images labtm 2.0 software system

After the addition of LDS Sample buffer and reducing agent (Nupage, Invitrogen, France) to GC samples and incubation at 95 °C for 10 min, each sample (containing 30 μg of total protein per well) was applied onto 4-12 % polyacrylamide gradient gels (Nupage, Invitrogen, France) inserted in a X-Cell SureLock system (Invitrogen, France) and then blotted to nitrocellulose membranes (Protan, Schleicher and Schuell, Dassel, Germany). The membrane was saturated during one hour with Phosphate Buffered Saline (PBS), 0.1 % Tween (PBS-T 0.1 %) and 5 % low-fat dried milk. An incubation was performed overnight at 4 °C, with a rabbit polyclonal human anti-ApoB100 antibody (H-300) (1:1000, sc- 25542, Biotechnology, Santa Cruz, USA), in PBS-T 0.1 %. Then, the membrane was incubated for one hour with an anti-rabbit HRP antibody (Biotechnology, Santa Cruz, USA) diluted in PBS-T 0.1 % (1:5000). APOB was detected using Supersignal West Pico Trial kit (Thermoscientific, Rockford, USA) and ChemiDocTM XRS Embellish-Images LabTM 2.0 Software system (Bio-Rad).

Culture medium samples (300 μl) were applied onto nitrocellulose membrane (Protan, Schleicher and Schuell, Dassel, Germany) and the membrane was washed with Tris Buffer Saline (TBS) with 0.1 % of Tween-20 (TBS-T 0.1 %), then saturated in TBS-T 0.1 % containing 5 % low-fat dried milk for one hour. The incubations were performed as described for western blots. Slot blots were detected using the Supersignal West Pico Trial kit (Thermoscientific, Rockford, USA) and analyzed using the ChemiDocTM XRS Embellish-Images LabTM 2.0 Software system (Bio-Rad).