IsolationofDL(LowDensityLipoprotein)FromHumanPlasma

This procedure involves use of human plasma, a potentially dangerous source of blood-borne disease. Wear long-cuffed gloves, and eye-protection.

Day one:

-Human Plasma is purchased from the Red-Cross. It should be 3-4 days old, from testing. The plasma bag is emptied by cutting the long tube with a pair of sharp scissors(be careful not to splash). To the plasma (300mL) is added 3mL of 100mM EDTA (sterile filtered) to a final concentration of 1mM. Protease Inhibitors may also be added as preservative.

-Plasma (~300mL) is spun at 12C@20,000 RPM for 20min in a TI-70 Rotor.   The tubes are removed and the chylomicrons are discarded.  If the plasma donor fasted (as instructed) there should be little cyhlomicrons in the upper fraction.  Discard the upper white, chylomicrons and transfer the  samples to new tubes.

-Plasma(~250ml) is spun at  12C@52,000 RPM for 24h in TI-70-Rotor. ~ 25mL per tube. Tubes using an inverted plug are used, with a red, aluminum screw-cap. Tubes are sealed and placed in Ultra Centrifuge.

Day Two :

-Mix the lower layer, leaving the greenish-pellet intact. The pellet contains glycogen and fibrinogen, and some LDL, but not enough to worry about.  Tubes may be inverted and squeezed to remove the contents.  Discard pellet, and tubes.

-Adjust the density of the LDL-Plasma to 1.06 using Potassium Bromide (KBr). If density is not adjusted LDL will not spin down. Calibrate pipette to ensure accuracy, using dH₂O.

-Add .075gKBr/mL Plasma.

-Check density weighing 1mL of solution. If not high enough, add KBr.

-Spin at 12C@40,000 RPM for 48h in TI-70 Rotor.

-Stop the rotor, without using the brake.

-Collect the LDL (uppermost fraction) taking care not to mix the layers or fractions.

-The LDL should be kept under nitrogen, dark and at 4C until use.  (This preparation according to other sources is good for up to 2 weeks.)