Density Gradient Media – News and Views

Lipid-rich plasma membrane domains have commonly been isolated from tissues and cultured cells as detergent-resistant membranes (DRMs), principally by flotation from a dense solution through a discontinuous or continuous density gradient containing a non-ionic detergent, usually Triton X-100 (TX100), but sometimes CHAPS. The resolution of DRMs is based on their low buoyant density and a flotation strategy is regarded as the best method for resolving any minor low-density fraction from predominantly denser material. Sucrose gradients were originally used in the separations but the much lower viscosity of Nycodenz solutions, compared to those of sucrose, allowed the centrifugation time at 200,000 g to be reduced from 18 h to 4 h. Although iodixanol gradients are slightly more viscous than the corresponding Nycodenz ones, the ease of preparation of gradient solutions from a commercial 60% (w/v) solution (OptiPrep) has made use of this medium very popular.

 An alternative approach, which avoids the use of a detergent, also uses an iodixanol gradient to resolve the low-density lipid-rich plasma membrane domains.

 Click HERE to access an Axis-Shield Mini-review of the detergent-based methods. It contains a brief methodological survey of the technique and a bibliography of all of the current papers (up to late 2009) that have reported the use of an iodixanol gradient to purify and analyze the lipid rafts.

 Click HERE to access a detailed protocol of the detergent-based method.

 Click HERE to access a detailed protocol of the detergent-free method.

Written by JohnG
Filed under: Membranes