Isolation of complexes from serum

Complexes can be isolated from serum by centrifugation on either a sucrose or Percoll gradient. The sucrose gradient is the easier of the two to set up.

 Sucrose gradient:

Place 5 ml of 50% sucrose in the bottom of a polyallomer 15 ml ultracentrifuge tube. Mark the top of this layer with a marker pen. Carefully load 3 ml of 30% sucrose onto the bottom layer of sucrose Carefully add 5 ml water onto the 30% sucrose layer Add 1 ml of serum (containing the complexes to be isolated) on the top of the gradient. The serum should sink to the interface of the water/30% sucrose layers. Centrifuge at 30 000 rpm in the ultracentifuge using a spin-out rotor for 15 hours at 250 C to pellet complexes at bottom of tube After centrifugation, freeze the tubes in liquid nitrogen until solid. Using a razor blade, cut off the bottom of the tube approximately a centimeter below the mark. Discard the rest of the gradient Allow the sucrose from the bottom of the tube to thaw, then pour off Resuspend pelleted complexes in 300 m l of water or Laemlli buffer (if to be run on a PAGE gel).

Isolating complexes from a Percoll gradient requires that an optimal gradient be determined for each complex to be isolated. For example, pLL/DNA complexes (pLL Mw 24,000) centrifuge into a Percoll layer of 1.04 g/ml. The best separation of serum from complexes was found using a gradient consisting of 2 mls of water, 3 mls 1.01 g/ml Percoll, 3 mls 1.02 g/ml, 2 mls 1.03 g/ml, 1 ml 1.04 g/ml, 1 ml 1.06 g/ml and 1 ml of 1.1 g/ml. Other complexes required different gradients for optimal separation of complexes from serum.