Nupage novex tris acetate mini gels

Proteins were extracted from MSCs/scaffold (n = 4) at the end of 1 and 2 weeks with pepsin (200 μg/mL in 0.08 M acetic acid, Sigma) for 72 hours at 4°C. The pepsin was subsequently inactivated with 1 M NaOH. The extract was concentrated using a Nanosep 30 centrifugal filter (30,000 Mw cutoff, Pall Life Sciences, USA). Samples were then separated by electrophoresis in NuPAGE Novex Tris-acetate mini gels (Invitrogen, USA) and electrophoretically transferred to a supported nitrocellulose membrane (Biorad Laboratories). The membranes were tested using western blot kit (Invitrogen, USA) according to the manufacturer's instructions. A brief description was as follows: the membranes were blocked with buffer for 1 hour and incubated overnight at 4°C with monoclonal antibodies against collagen II and TGF-β3 diluted to 1 : 500 in blocking buffer. The membranes were then washed five times with washing buffer and incubated for 30 minutes with secondary antibodies diluted to 1 : 200 in blocking buffer. The membranes were rinsed with washing buffer again and incubated with ECL working solution for 5 minutes. The signal was detected using VersaDoc Imaging System (Biorad Laboratories) and relative intensities of positive bands were compared between groups.