Benzonase® Nuclease Q&A
Benzonase® Nuclease is used in applications that require complete digestion of nucleic acids. It is commonly used to remove contaminating host-cell DNA and RNA from recombinant protein preparations, reduces viscosity in protein extracts, prevents cell clumping, and can eliminate protein-nucleic acid interactions that may interfere with experiments.
For what purposes would I want to use Benzonase® Nuclease?
Applications include: reduction of viscosity to facilitate processing (e.g., recombinant protein purification, viscosity-sensitive applications using mammalian cell lysates, etc.); reduction of clumping in stored peripheral blood mononuclear cell (PBMC) samples prior to processing; preparation of inclusion bodies to allow successful protein renaturation; and removal of negatively charged nucleic acids from samples prior to two-dimensional SDS-PAGE.
How can Benzonase® Nuclease be inactivated? How can it be removed?
Reversible inhibition can be achieved using EDTA to chelate essential metal ions. Irreversible inactivation can only be accomplished with extreme conditions (100 mM NaOH at 70 °C for 30 minutes). Benzonase® Nuclease can be separated from the target product using chromatography.
My Benzonase® Nuclease was left out on the bench. Is it still good?
We have done extensive stability testing on Benzonase® Nuclease, and find that it is extremely stable. Even with extended incubations at 37 °C, Benzonase® Nuclease maintained > 90% activity for several months. Recommended storage temperature is -15°C to -25°C. Do not store <-25°C.
I want to use a different buffer. What conditions are absolutely required for full activity of Benzonase® Nuclease? What will reduce its activity?
Benzonase® Nuclease requires 1‒2 mM Mg2+ for activity. Benzonase® Nuclease is inhibited (approximately 50% activity) by monovalent cation concentrations >150 mM, phosphate concentrations >20 mM, and by ammonium sulfate concentrations >25 mM.
Is Benzonase® Nuclease compatible with protease inhibitor cocktails?
Yes. However, caution should be exercised, since many protease inhibitor cocktails include EDTA. Concentrations of greater than 1 mM EDTA will inhibit Benzonase® activity.
My protein is insoluble and I need to perform purification under denaturing conditions. Will Benzonase® Nuclease still work in urea?
Benzonase® Nuclease activity actually increases in presence of urea at concentrations up to 6 M. At 6 M urea, enzyme activity first increases, then decreases over time. At 7 M urea, Benzonase® Nuclease denatures after 15 minutes, and activity is lost. However, significant degradation of nucleic acids occurs before inactivation. Higher initial concentrations of Benzonase® Nuclease can partially compensate the effects of 7 M urea.
Why do you have so many varieties of Benzonase® Nuclease? What does HC mean? What impact does 90% versus 99% purity have?
To meet the widest possible range of processing and cost requirements, Benzonase® Nuclease is available in two different purity grades: Purity grade I (>99% pure) and Purity grade II (>90% pure). Both grades are available at 25 U/μL or at a high concentration (HC), which is defined as 250 U/μL. For bulk purchases, contact Custom Services.
What is the end result of complete nucleic acid degradation by Benzonase® Nuclease?
Benzonase® Nuclease completely digests nucleic acids to 5’-monophosphate terminated oligonucleotides that are 3‒5 bases in length.
My process uses high amounts of salt. Do you have a salt-tolerant Benzonase® Nuclease?
Yes, we have Benzonase® Salt Tolerant Endonuclease. It operates under the same conditions as standard Benzonase® products, with one important exception: monovalent cation concentration. A minimum of 200 mM NaCl is recommended.
The enzyme is functional between pH 6 and 10 and from 0 °C to above 37 °C. Mg2+ at 1–2 mM is required for activity.