Separation of primary nuclear membrane

Separation of primary nuclear membrane

Reagents and equipment:
1. DNase I;
2. KCl (1 mol / L dissolved in 20 mmol / L Tris-HCl, pH 7.4);
3. MgCl2 (1 mol / L stock solution);
4. Phenylmethylsulfonyl fluoride (PMSF) (1 mol/L stock solution prepared from absolute ethanol);
5. Purified nuclei;
6. Buffer: 2mmol / L Tris-HCl, pH 7.4;
7. Membrane suspension buffer (ESB): 0.25 mol / L sucrose, 2 mmol / L Tris-HCl, pH 7.4;
8. Gradient solution: 1.0mol / L, 1.5mol / L, 1.8mol / L, 2.0mol / L sucrose dissolved in 2mmol / L Tris-HCl, pH 7.4;
9. All solutions except MgCl2 must contain 0.1 mmol/L phenylmethylsulfonyl fluoride;
10. Abbé refractometer;
11. High speed centrifuge with fixed angle rotor and centrifuge tube;
12. Ultracentrifuge with fixed angle and bucket rotor and centrifuge tube;
13. Phase contrast microscope;
14. Ultraviolet spectrophotometer (with quartz cuvette);

experimental method:
All operations were carried out at 0-4 °C unless otherwise noted.
1. The purified primary cell nuclei were immersed in buffer for 10 min to inflate to decondense. After centrifugation for 10 min at about 10000 g, resuspend the pellet with buffer and repeat this step twice;
2. Resuspend the gel-like swollen nuclei pellet in buffer containing DNase I (0.01 g/L). Mix well and incubate for 30 min at room temperature. The supernatant is adjusted to contain about 0.1 mmol/L MgCl 2 in the middle of the incubation. As the DNA is cleaved by cleavage, the chromatin quickly loses its gel-like properties. The production of "shadow" of the nuclear membrane can be detected by phase contrast microscopy;
3. Using a fixed angle rotor or a bucket rotor, centrifuged at about 60,000g for 30min;
4. Resuspend the crude nuclear membrane in buffer and centrifuge again. Repeat this step until the DNA is released (ie, monitor the supernatant for absorbance at 280 nm until a very low platform is reached);
5. Selectively wash with a KCl solution to obtain a nuclear membrane to accelerate the removal of ion-binding proteins, especially histones. Then centrifuged at about 60,000 g for 30 min;
6. Resuspend the nuclear membrane with the nuclear membrane suspension buffer, cover the prepared four gradient solutions, and centrifuge in a bucket centrifuge for several hours or overnight to collect the nuclear membrane equal density layer;
7. The purified nuclear membrane should mainly accumulate at the interface of 1.5mol/L and 1.8mol/L two sucrose layers, and carefully remove the nuclear membrane layer from the gradient liquid with a Pasteur pipette;
8. Check the integrity of the nuclear membrane by phase contrast microscopy or by negative staining and/or thin-film electron microscopy with agarose embedding;
9. Perform necessary biochemical analysis on purified specimens, such as SDS-PAGE or enzymatic experiments; endogenous peroxidase is a suitable marker for rat liver nuclear membrane, as well as Mg-ATPase and many Quality net enzyme

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