Purification of nuclei from tissues and cells in isoosmotic iodixanol gradients – a methodological review

The low-speed pellet produced by the centrifugation of tissues or cell homogenates at 600-1000 g for 10 min will contain 90-95% of the total nuclei, but it will be contaminated by significant amounts of mitochondria, large fragments of membrane, intact or partially broken cells and other organelles trapped by these rapidly sedimenting particles.

 Although the contamination may be partially reduced by repeated washing of the pellet, this procedure risks progressive damage to the nuclei by the shearing forces used to disperse the pellet. In the nineteen-sixties two influential papers [1,2] were published both of which reported the pelleting of nuclei through high-density sucrose solutions to remove the contamination effectively.

 In the method of Widnell and Tata [1] a crude nuclear pellet (homogenate centrifuged at 600 g for 10 min) was washed once in the homogenization medium; suspended in 2.2 M sucrose, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.4 and then the nuclei pelleted at 60-80,000 g for 80 min at 4°C. 

The method of Blobel and Potter [2] omitted the low-speed pelleting of the nuclei directly from the homogenate: the sucrose concentration in the filtered homogenate was adjusted to 1.6 M by addition of 2.3 M sucrose, 50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, and then layered over this 2.3 M sucrose solution. The nuclei were pelleted at 130,000 g for 30 min at 4°C.