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Here are answers to the most frequently asked questions about Benzonase® endonuclease
What type of nucleic acids does Benzonase® endonuclease work on? Can I use it when isolating RNA?
Benzonase® endonuclease is a promiscuous endonuclease that attacks and degrades all forms of DNA and RNA (single-stranded, double-stranded, linear and circular).
For what purposes would I want to use Benzonase® endonuclease?
This question cannot be answered in general. For the case at hand, the optimum conditions have to be determined experimentally. There are several parameters that influence the activity of Benzonase® endonuclease. Therefore, the optimum conditions will vary from process to process and need to be determined experimentally.
Applications include: reduction of viscosity to facilitate subsequent processing (e.g. recombinant protein purification, viscosity-sensitive applications for mammalian cell lysates, etc.); reduction of clumping in stored peripheral blood mononuclear cell (PBMC) samples prior to processing (see Perrin 2008 inNovations 28, 21); preparation of inclusion bodies to allow successful renaturation of protein, removal of negatively charged nucleic acids from samples prior to two-dimensional SDS-PAGE, adenovirus purification, monoclonal antibody production, large-scale purification of lentiviral vectors, etc.
How can Benzonase® endonuclease be inactivated? How can it be removed?
Irreversible inactivation can only be accomplished under extreme conditions (100 mM NaOH at 70C for 30 minutes). However, the conditions that are needed to denature proteins may be harmful to your end product. Reversible inhibition can be achieved by complexation of Mg2+ ions with EDTA and / or phosphate.
Benzonase® endonuclease can be separated / removed from the target product during downstream processing using chromatographic separation methods. Depending on the characteristics of the desired protein, ion exchange, hydrophobic interaction, hydroxylapatite or gel permeation chromatography can be used. Assuming a significant difference in molecular size between Benzonase® endonuclease and the target protein, ultrafiltration may be used successfully. Benzonase® endonuclease is not or only weakly bound to anion exchange resins under a variety of conditions: pH 7.0 – 9.0 at 50 mM NaCl. Benzonase® endonuclease elutes from cation exchange resins below 200 mM NaCl at pH 6.0 and is not bound to weak cation exchange resin at pH 6.0
It is recommended to optimize the separation protocol in every special case using the above-mentioned results as a guideline.
Do you offer an immobilized Benzonase® endonuclease?
No. All efforts to bind Benzonase® endonuclease to a support that meets the demands of a commercial product with respect to activity, stability and regulatory requirements have so far been unsuccessful. But, we do not give up.
Is Benzonase® endonuclease free of protease activity?
Yes, it is supplied without detectable protease activity and is thus not degraded during its “work”.
Is Benzonase® endonuclease 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® endonuclease activity.
My Benzonase® endonuclease was left out on the bench. Is it still good?
We have done extensive stability testing on Benzonase® endonuclease, and found that it is extremely stable. Even with extended incubations at 37C, Benzonase® endonuclease maintained > 90% activity for several months.
I want to use a different buffer. What conditions are absolutely required for full activity of Benzonase® endonuclease? What will reduce its activity?
Benzonase® endonuclease requires 1-2 mM Mg2+ for activity. Benzonase is inhibited (approximately 50% activity) by monovalent cation concentrations >50%, phosphate concentrations >20 mM and ammonium sulfate concentrations >25 mM.
My protein is insoluble and I need to perform purification under denaturing conditions. Will Benzonase® endonuclease still work in urea?
Benzonase® endonuclease activity actually increases in the presence of urea up to 6 M.
At 6 M urea, enzyme activity first increases, then decreases over time. At 7 M urea, Benzonase® endonuclease denatures after 15 minutes, and activity is lost. However, significant degradation of nucleic acids occurs before inactivation. Higher initial concentrations of Benzonase® endonuclease can partially compensate the effects of 7 M urea.
Why do you have Benzonase® endonuclease in two different purity grades? What impact does 90% versus 99% purity have?
To meet the widest possible range of processing and cost requirements, Benzonase® endonuclease 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 that is defined as 250 U/µl.
What is the end result of complete nucleic acid degradation by Benzonase® endonuclease?
Benzonase® endonuclease completely digests nucleic acids to 5’-monophosphate terminated oligonucleotides 2-5 bases in length.
At which step do I have to introduce Benzonase® endonuclease in my process?
The answer to this question will vary depending on why you are using Benzonase® endonuclease. However, as a general rule, Benzonase® endonuclease is usually best added after the fermentation step and before the capture step.
Do you offer an ELISA kit for the assay of traces of Benzonase® endonuclease?
Yes. An ELISA kit is available.
Would you like more information? Please contact us.
