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:

1. 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.
2. Carefully load 3 ml of 30% sucrose onto the bottom layer of sucrose
3. Carefully add 5 ml water onto the 30% sucrose layer
4. 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.
5. 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
6. After centrifugation, freeze the tubes in liquid nitrogen until solid.
7. Using a razor blade, cut off the bottom of the tube approximately a centimeter below the mark.
8. Discard the rest of the gradient
9. Allow the sucrose from the bottom of the tube to thaw, then pour off
10. 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.