Procedure [6265

Except where indicated carry out all operations at 0-4 °C.

1. Swell/decondense nuclear chromatin by incubation of purified nuclei in buffer for 10 min, followed by centrifugation at ~ 10 000g for 10 min. Resuspend the pellet in buffer and repeat twice. (!)

2. Resuspend the gelatinous pellet of swollen nuclei in buffer containing deoxyribonuclease I (10 ^g/ml). Incubate at room temperature for 30 min with mixing. Halfway through the incubation adjust the suspension to ~0.1 mM MgCl2. The gelatinous nature of the chromatin should be rapidly lost as DNA cleavage occurs. Monitor the production of nuclear 'ghosts' by phase contrast microscopy. ©

3. Centrifuge at ~60000g for 30 min in a fixed-angle or swinging-bucket rotor.

4. Resuspend the pellet of crude nuclear envelopes in buffer and centrifuge again. Repeat this step several times until the release of DNA, monitored by the absorbance of the supernatant at 280 nm, has reached a low plateau. ©

5. Optionally, wash the nuclear envelope with the KCl solution to assist the removal of ionically bound proteins, histones in particular, followed by centrifugation at ~60 000g for 30 min. ©

6. Resuspend the washed nuclear envelope in ESB and overlay on a step gradient made from the four gradient solutions, in a swinging-bucket rotor. Centrifuge for several hours (or overnight) to band the nuclear envelope isopycnically. ©5 ©6

7. The purified envelopes should band predominantly at the 1.5 M/1.8 M sucrose interface. Carefully remove the nuclear envelope layer from the gradient using a Pasteur pipette.

8. Monitor the integrity of the nuclear envelope by phase contrast microscopy and by negative staining and/or thin section electron microscopy using the agarose embedment procedure (Protocol 2.8). ©

9. Perform the required biochemical analysis on the purified preparation, for example SDS-PAGE and enzymology; endogenous peroxidase is a reasonable marker enzyme for the nuclear envelope from rat liver, Mg-ATPase and many ER enzymes are also present [66].

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