Reagents and equipment:
Heparin
2. Phenylmethylsulfonyl fluoride (PMSF) (dissolved in ethanol to make a 1 mol / L stock solution);
3. Purified nuclear membrane;
4. extraction buffer (EB): 1% (volume fraction) 10mmol / L Tris-HCl Triton X-100 is, (pH7.4), 0.1mmol / L phenylmethylsulfonyl fluoride;
5. Tris Buffer: 2mmol / L Tris-HCl, pH7.4,10mmol / L Tris-HCl, pH7.4;
6. gradient solution: 0.25mol / L sucrose solution and 1.5mol / L sucrose solution was dissolved in 10mmol / L Tris-HCl, pH7.4;
7. All solutions must contain 0.1mmol / L phenylmethylsulfonyl fluoride;
8. gradient collector;
9. The high-speed centrifuge, and with fixed-angle rotor centrifuge tube;
10. ultracentrifuge it equipped with a fixed angle and swinging bucket rotor, centrifuge tubes;
11. Ultrasonic generator (probe type with timer);
12. Dual chamber gradiometer or Gradient MasterTM;

experimental method:
1. Using the extraction buffer, extract the primary nuclear membrane at 4 ° C for 30 min;
2. Centrifuge at 20000g for 30 min to loosely precipitate the core pore complex sheet. The pellet was carefully resuspended in 2 mmol/L Tris-HCl, (pH 7.4) and centrifuged again;
3. Resuspend the pellet (eg 1 ml) in small volumes with 10 mmol/L Tris-HCl (pH 7.4) and sonicate on an ice bath. In order to prevent the sample from producing heat, the ultrasonic process is repeated for a short time (such as 10s), and the interval is cooled;
4. By preparing negatively stained specimens, such as 20g/L ammonium molybdate (pH7.0) or 50g/L ammonium molybdate-1g/L trehalose (pH7.0) for electron microscopy analysis, timely monitoring of nuclear membrane rupture and Release of the nuclear pore complex. If necessary, continue with ultrasound treatment;
5. The sample is applied to a linear density gradient solution of 0.25-1.5 mol/L sucrose-10 mmol/L Tris-HCl (pH 7.4), and centrifuged at 4 ° C, about 60,000 g for 1-2 h;
6. Partially collected from a sucrose gradient solution;
7. The electron microscopy using negative staining ingredient content were collected to determine the nuclear pore complex precipitation in a sucrose gradient solutions position, and integrity analysis;
8. constituents gradient SDS-PAGE, separated proteins detected situation;

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 Specifications

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