Tln100 rotor

Binding reactions (1.0-ml volume), each containing 500 μg of LDL and 1 μg of PCSK9 (in concentrated conditioned media) and 0.5% BSA (w/v) in HBS-C buffer (25 mm HEPES-KOH, pH 7.4, 150 mm NaCl, 2 mm CaCl₂) were incubated at 37 °C for 1 h. LDL-bound and free PCSK9 were then separated by Optiprep gradient ultracentrifugation according to a modified version of a protocol described previously (73 (link)). Briefly, a 9% Optiprep sample solution was prepared by diluting 1.0 ml of each binding reaction with 0.45 ml of 60% Optiprep and 1.55 ml of 25 mm HEPES-KOH, pH 7.4. The 9% sample was overlaid with 25 mm HEPES-KOH, pH 7.4, in a 3.3-ml Optiseal tube (Beckman). Tubes were centrifuged in a TLN100 rotor (Beckman Coulter) at 100,000 rpm for 2 h at 4 °C. LDL-containing fractions (600 μl) were collected by tube puncture into a 1-ml syringe. The entire LDL fraction was immunoprecipitated for 18 h at 4 °C using anti-PCSK9 rabbit polyclonal 1697 (see above) and anti-rabbit Trueblot agarose beads (Rockland). The collected beads were washed, and immunoprecipitated proteins were resolved by SDS-PAGE on 8% acrylamide gels. PCSK9 content was then quantified by Western blotting, and values were normalized to reactions containing WT PCSK9.

DNA was separated by density centrifugation by adding 800–1000 ng DNA to a solution containing 2.55 mL saturated (1.9 g mL⁻¹) cesium chloride, 450 µL gradient buffer (200 mM trisaminomethane, pH 8, 200 mM potassium chloride, 2 mM Ethylenediaminetetraacetic acid), and ~200 µL of TE buffer in 3.3 mL OptiSeal ultracentrifuge tubes (Beckman Coulter). Ultracentrifuge tubes were balanced in a Beckman TLN-100 rotor and spun at 127,000 × g for 96 h using an OptimaMAX TL ultracentrifuge.
The resulting cesium chloride gradient was fractionated into approximately 14, 150–200 µL fractions. Fraction density was measured using a digital refractometer (Reichert), then purified by using an isopropanol precipitation method and re-suspended in 50 µL TE buffer. DNA concentrations in each fraction were measured with the Qubit BR dsDNA assay (Invitrogen).