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.
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
D016475:3T3 Cells / Q000201:enzymology; D000818:Animals; D001426:Bacterial Proteins / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005615:Freezing; D008274:Magnesium / Q000494:pharmacology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D001616:beta-Galactosidase / Q000187:drug effects* / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D001616:beta-Galactosidase; D008274:Magnesium; D002118:Calcium