Homogenization buffer B

0.2 mM EDTA

10% glycerol (v/v) (or 0.25 M sucrose)

5 mM magnesium acetate

50 mM Tris-Cl (pH 7.5)

Just before use, add to the chilled buffer with adequate stirring:

0.5 mM DTT (dithiothreitol)

Dilute from a 0.5 M stock solution in cold H₂O. Store stock solution frozen.

1.0 mM PMSF (phenylmethylsulfonyl fluoride)

Prepare a 0.2M stock solution in 2-propanol; the reagent will crystallize from 2-propanol when stored at −20ºC.

AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride) is a water-soluble alternative to PMSF that can be used at the same molar concentration (0.1-1.0 mM) for most applications.