All reagents and buffers for microsomes and SRP preps should be made in sterile/baked glassware with water straight from the purifier, and stored in the cold room. 10 x dog
2 M sucrose - use 'RNA use only' container!
171.15 g/250 mL
2 M triethanolamine (TEA) (pH 7.5)
Adjust pH with glacial acetic acid, use nuclease free pH meter
0.1% SDS - Dilute from the 20% stock. Use this to read the OD of your microsomes.
PMSF (phenylmethyl sulfonyl fluoride)
Make fresh,
DTT (dithiothreitol)
1M DTT is 154 mg in 1 mL dH2O. Store at -20oC. DTT preserves the natural inhibitor of ribonuclease that is present in dog pancreas. It is because of this inhibitor that we use pancreas from dog (rather than some other animal) to make microsomes.
Use both PMSF and DTT at 1 mM final in all buffers that come into contact with the homogenate.