Cnbr activated sepharose 4b resin

A rHDL-containing apoA-I and cholesteryl oleate were synthesized in accordance with the method described by Cho (25 (link)) and Cho et al. (26 (link)) using trace amounts of [³H]-cholesteryl oleate (TRK886, 3.5 μCi/mg of apoA-I; GE Healthcare). The rHDL was immobilized using CNBr-activated Sepharose 4B resin (Amersham Biosciences) for easy separation after the reaction in accordance with the manufacturer's instructions. CE transfer reaction was performed in 300-μL reaction mixtures containing human serum (20 μL) or HDL₃ (20 μL, 2 mg/mL) as a CETP source, [³H]-rHDL-agarose (20 μL, 0.25 mg/mL) as a CE-donor, and human LDL (20 μL, 0.25 mg/mL) as a CE-acceptor. After incubation of 4 h at 37°C, the reaction was halted via brief centrifugation (10,000 g) for 3 min at 4°C. The supernatant containing CE-acceptor (150 μL) was then subjected to scintillation counting, and percentage transfer of [³H]-CE from [³H]-rHDL to LDL was calculated.

A rHDL-containing apoA-I and cholesteryl oleate was synthesized in accordance with the method described by Matz and Jonas [25 (link)], modified by Cho [26 (link)] using trace amounts of [³H]-cholesteryl oleate (TRK886, 3.5 μCi/mg of apoA-I; GE Healthcare). The rHDL was immobilized using CNBr-activated Sepharose 4B resin (Amersham Biosciences) for easy separation of rHDL after the reaction, in accordance with the manufacturer’s instructions. CE transfer reaction was performed in 300-μL reaction mixtures containing human plasma (20 μL) or HDL₃ (20 μL, 2 mg/mL) as a cholesteryl ester transfer protein (CETP), [³H]-rHDL-agarose (20 μL, 0.25 mg/mL) as a CE-donor, and human LDL (20 μL, 0.25 mg/mL) as a CE-acceptor source, as described previously [27 (link)]. After incubation at 37°C with shaking around 200 rpm, the reaction was halted via brief centrifugation (10,000g) for 3 min at 4°C to sediment [³H]-rHDL-agarose. Supernatants containing CE-acceptor (150 μL) and the agarose pellet (50 μL) were then separately subjected to scintillation counting, and percentage transfer of [³H]-CE from [³H]-rHDL to LDL was calculated. For non-specific reactions, a parallel experiment was simultaneously carried out in the control tube, which includes all constituents except the CETP source.