Western Blot Analysis of Protein Purification

Protein was purified by acetone precipitation from the cell lysates as previously described [29 (link)]. Briefly, the protein pellet was dissolved in 1% SDS buffer, and centrifuged for 5min at 14000 rpm. Proteins were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane followed by Western blot. 8% non-fat milk in TBS containing 0.1% Tween-20 was used to block non-specific binding. The blot was then incubated with an anti-TNF-α rabbit monoclonal antibody (1:200, Abcam, Cambridge, MA, USA), or an anti-β-actin rabbit monoclonal antibody (1:200, Abcam) at 4°C overnight followed by a secondary antibody (peroxidase-conjugated anti-rabbit IgG 1:5000, Millipore Corporation). An Azure C300 chemiluminescence system (Dublin, CA, USA) was used for detection.