Chapter 13 Large-Scale Isolation of Nuclear Membranes from Bovine Liver

This chapter describes the procedure for the isolation of nuclear membranes from bovine liver which is the largest preparation reported to date, making it extremely useful for nuclear membrane fractionation studies. The size of the preparation can be scaled up or down according to the needs and facilities available. A beef liver weighing 10–15 pounds is obtained within an hour after removal from the animal. Excess blood is removed by washing with 0.25 M sucrose TKM. The surrounding membrane is then peeled from the surface of the liver. Crude nuclei pellets are isolated by differential centrifugation using the 4-liter capacity swinging-bucket rotor of the International PR-2 Centrifuge at 800 g for 20 minutes. Nuclei are purified by centrifuging through a dense sucrose solution. The crude nuclear pellets are resuspended in 2.3 M sucrose TKM by vigorously shaking, and filtered through 8 layers of cheese cloth. The nuclear preparation was analyzed by light, Nomarski interference, and electron microscopy. Nuclear pore complexes are an excellent ultrastructural marker for identifying nuclear membranes, if the membranes are isolated in an intact enough state to demonstrate pore complexes.