Table 5.
Recipe for 10 % and 30 % sucrose solutions to prepare sucrose gradients. The recipe indicates the volumes required for two samples. Scale-up as necessary.
| Gradient Buffer | ||||
|---|---|---|---|---|
| 10 % sucrose | 30 % sucrose | |||
| Reagent | [Stock] | [Final] |
2x Volume
(5 mL) |
2x Volume
(5 mL) |
| Tris pH 7.4 | 1 M | 10 mM | 50 μL | 50 μL |
| NH4Cl | 1 M | 100 mM | 500 μL | 500 μL |
| MgCl2 | 1 M | 10 mM | 50 μL | 50 μL |
| PMSF | 100 mM | 1 mM | 50 μL | 50 μL |
| Digitonin (high purity) | 10 % | 0.05 % | 25 μL | 25 μL |
| Yeast protease inhibitor cocktail | 100x | 1x | 50 μL | 50 μL |
| RNase inhibitor | 40 U/μL | 0.1 U/μL | 12.5 μL | 12.5 μL |
| Sucrose | 50 % | 10 %/30 % | 1000 μL | 3000 μL |
| H2O | - | - | 3262.5 μL | 1262.5 μL |