MAGNESIUM CHELATION INACTIVATES BETA-GALACTOSIDASE IN 20-DEGREES-C STORAGE

Mammalian cell lysate containing beta-galactosidase (beta-Gal) derived from the transient expression of the bacterial lacZ gene driven by the human beta-actin promoter loses activity progressively over time in storage at -20-degrees-C in the presence of EDTA. The simultaneous presence of NaCl with EDTA exacerbates such an inactivation, although NaCl by itself does not. However, EGTA, a chelating agent that preferentially binds Ca2+ over Mg2+, does not inactivate beta-Gal. Addition of equal or higher molar concentration of Mg2+ (as MgCl2) or Ca2+ (as CaCl2), both effectively chelated by EDTA, to an EDTA-containing lysate prevents this cold-related inactivation, but does not reactivate the enzyme. Therefore, the chelation of Mg2+ by EDTA at -20-degrees-C inactivates beta-Gal. Storage of cell lysate at -70-degrees-C completely prevents the EDTA-induced inactivation of beta-Gal. It is recommended that when beta-Gal activity is used as the reporter for gene expression 1) EDTA should not be used to prepare cell lysate and 2) the cell lysate should be stored in a -70-degrees-C freezer to preserve full activity.