TABLE 1.
Preparation of HSS
| Purification steps | Product | |
|---|---|---|
| 1. | Remove the liver, immediately after killing by guillotine, between 7:00 and 8:00 am | |
| 2. | Mince and then homogenize the liver in 150 mM sodium acetate buffer, pH 4.65 (35:100 w/v) | |
| 3. | Ultracentrifuge homogenate at 24,000 g for 30 min at 4° C | Cytosol fraction (Cyt-F) |
| 4. | Heat at 65° C for 15 min | |
| 5. | Centrifuge at 30,000 g for 20 min at 4° C, collect supernatant and add to it 6 vol of cold ethanol (1:6, v/v) | |
| 6. | Stir at 2–8° C for 2 hr | |
| 7. | Centrifuge 30,000 g for 20 min at 4° C | |
| 8. | Resuspend precipitate in 0.150 mM ammonium acetate, pH = 6 | Alcohol fraction (OH-F) |
| 9. | Filter OH-F through an Amicon membrane with a molecular weight cutoff of 30,000 Da | |
| 10. | Collect the filtrate and concentrate it by a 500-Da cutoff Amicon membrane | Mr 30,000 fraction (30 kDa-F) |
| 11. | Lyophilize 30 kDa-F | |
| 12. | Resuspend lyophilized 30 kDa-F in phosphate buffer 5 mM, pH 6, and perform chromatography using mono Q HR 5/5 column with a linear 0–200 mM NaCl gradient in phosphate buffer | |
| 13. | Collect the chromatographic peak at 150 mM NaCl gradient | 150 fraction (F150) |