| | Re: Lipoprotein preparation
Selective isolation of the LDLs: mix at the vortex, 50 - 100 µl of serum/plasma with 1.0 - 2.0 ml of selective precipitating reagent, amphophillic polymers, for the LDLs (dissolve in imidazole buffer 25 mM at pH 6.1, 0.4 mg/ml anionic polycyclic surfactant activator, 0.6 - 0.8 mg/ml anionic polycondensated surfactat activator and 12.4 mM polysubstituted dioxan), and incubate at 4 °C for 30 min. Centrifuge at 4000 rpm (2800 g) for 5 min., discard the supernatant and pellet is resuspend in 0.5 ml of resuspending solution (0.15 M trisodium citrate and 0.11 M NaCl). For a greater purity the precipitate must be once washed with selective precipitating reagent and then centrifuge at 4000 rpms for 5 min. Suspend pellet in solubilizing solution (0.01% Triton X-100 in 100 ml of aqueous 50 g/L of NaCl) kept at 37 °C, and to mix at the vortex for resuspending the LDLs. Dialyze the solution against repeated change of dialyzing solution (phosphate buffer 0.02 M at pH 7.4, 0.15 M NaCl, 0.2 g/L NaN3 and 0.1 g/L Na2EDTA). The liquid of dialysis could be also formulated as 8.4 mM K2HPO4, 1.6 mM KH2PO4, 1.34 mM EDTA and 0.65 mM of glutathione (EDTA and glutathione are added for inhibiting lipoperoxidation).