Table 6.
Polysome lysis buffer prepared fresh
| Component | Final Concentration |
|---|---|
| Tris-HCl at pH 7.5 | 10 mM |
| KCl | 100 mM |
| MgCl2 | 5 mM |
| Sodium deoxycholate | .5% w/ve |
| Nonidet-P40 | 1 % v/v |
| DTT | 10 mM |
| cycloheximide | 150 μg/mL |
| SUPERase In RNase Inhibitor | 1 U/μL |
| Protease inhibitor cocktail | -- |
| Molecular Grade H2O | -- |
This must be prepared the day of the experiment. Each sample will require 1 mL and it is recommended to make an additional milliliter. To prepare 4 mL: thaw 4 aliquots of previously prepared lysis buffer stocks on ice. Combine 3.72 mL into a conical tube. Add 40 μL of Nonidet-P40, 150 μL of 1M DTT, 40 μL of 150 mg/mL cycloheximide in EtOH, and 40 μL of SUPERase•In RNase Inhibitor. Mix well by vortexing and keep on ice until ready for use. ‡Nonidet-P40 is no longer commercially available, and an alternative may need to be used. Final concentrations of detergent for lysis may need to be adjusted to compensate for this or according to your tissue of interest.