Tris glycine native buffer

BisTris NativePAGE running buffer and Tris-Glycine native buffer (Invitrogen) were prepared according to the manufacturer's instructions and 1% (v/v) NativePAGE cathode additive was added to BisTris NativePAGE buffer. Buffers were then chilled to 4°C. Samples were prepared by adding sample buffer to 1× and then separated on 4-16% NativePAGE Bis-Tris gels (Invitrogen) for 90 min at 150 V or on 4-12% Novex Tris-Glycine gels (Invitrogen) for 60 min at 125 V, on ice. Gels were either stained using Coomassie staining (0.02% R-250 (w/v) in 10% (v/v) acetic acid, 40% (v/v) methanol, then destained in 8% (v/v) acetic acid) or used for western blotting. Native gels were incubated in 2× transfer buffer with 10% methanol for 10 min and then transferred to 0.4 μm PVDF membranes using the iBLOT semi-dry blotter (7 min programme P0). Post-transfer, the membranes were incubated for 15 min in 8% acetic acid, rinsed in reverse osmosis H 2 O and then incubated in hot PBS for 10 min for antigen-retrieval. Membranes were dried, reactivated in methanol and stained using Ponceau S. The immunodetection procedure was the same as described for SDS-PAGE but the apoD C1 antibody was used at a 1:1,000 dilution.