Native page bistris gels system 4 16

We detected the interactions of rTCP96 and TLR ligands by BN-PAGE and Western blot analysis. rTCP96 (5 μm) was incubated with TLR ligands for 30 min at 37 °C. The samples were loaded under reducing conditions on BN-PAGE (Native PAGE BisTris Gels System 4–16%, Invitrogen) according to the manufacturer's instructions, which we followed by Western blotting. Although the complex formation is known to be a process involving several intermediate steps, this assay is accepted in the field as an approximate measure of the binding capability between peptides and ligands.

Male Sprague Dawley rats purchased from Taconic (Ry, Denmark) were used at the age of 10–11 weeks. Rats were on normal diets. Blood samples were collected and serum samples (200 μl) were mixed with apoA-I WT and A164S (0.75mg/ml). The mixtures were incubated for 2, 4 and 8 h at 37°C. The samples were separated by NativePAGE Bis-Tris Gels System 4–16% (Invitrogen), and transferred to PVDF membranes, probed with anti-human apoA-I antibodies (Abcam) and immune detection performed with HRP-conjugated secondary antibodies (GE Healthcare). Blots were imaged using the Odyssey Fc system (LI-COR) and quantified using Image studio v2.0 software. The animal procedures were approved by the Malmö/Lund Committee for Animal Experiment Ethics.