New England Biolabs, G8024L, NEBExpress® Salt Active Nuclease, GMP Grade

Related Categories Protein Tools,, DNA Modifying Enzymes & Cloning Technologies Applications Protein Purification,, Protein Analysis Tools,, Protein Expression Specification Unit Definition One unit is defined as the amount of enzyme required to release 32 pmol of FAM from FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 25°C in a 50 µL reaction in 25 mM Tris-HCl, 500 mM NaCl, 5 mM MgCl 2 , 0.005% Tween 20 (pH 8.5 @ 25°C). Storage Buffer 25 mM Tris-HCl 500 mM NaCl 5 mM MgCl2 50% Glycerol pH 7.5 @ 25°C FAQ Q: I see that NEB offers two versions of NEBExpress® Salt Active Nuclease. What is the difference between NEBExpress Salt Active Nuclease (NEB #M0764) and NEBExpress Salt Active Nuclease, GMP Grade (NEB #G8024)? A: NEBExpress Salt Active Nuclease, GMP Grade is manufactured according to New England Biolab’s GMP-grade* quality standards for stringent quality documentation and manufacturing needs, and includes additional testing such as endotoxin and bioburden testing. Both enzymes are enzymatically identical and formulated in the same buffer. However, NEBExpress Salt Active Nuclease, GMP Grade (NEB #G8024) is offered at a 10X higher concentration at 1,000,000 U/ml (1 mg/ml) to minimize dilution of your cell culture/sample compared to NEBExpress Salt Active Nuclease (NEB #M0764), which is offered at 100,000 U/ml. Q: Can I order NEBExpress® Salt Active Nuclease, GMP Grade (NEB #G8024) at a larger volume or at a different concentration? A: Yes. To order NEBExpress Salt Active Nuclease, GMP Grade (NEB #G8024) in a larger size or a customized format, contact our Customized Solutions team. Q: What are the benefits of working in a high salt buffer? A: Performing a purification process in high salt (≥ 200 mM NaCl) allows impurities (nucleic acids) to detach from proteins or viruses by disrupting electrostatic interactions between these molecules. High salt can also improve protein solubility when aggregation is due to electrostatic interactions. For these reasons, many bioprocessing methods call for high salt. NEBExpress® Salt Active Nuclease can be used directly in these conditions to further alleviate high viscosity challenges, reduce back pressure, and/or remove nucleic acid. Q: Does NEBExpress® Salt Active Nuclease have the same efficiency as Serratia marcescens nuclease? A: No. NEBExpress Salt Active Nuclease is more active (about 4x, when normalized at the same concentration) than Serratia marcescens nuclease on DNA and RNA when used in a buffer with NaCl/KCl ≥ 300 mM. Q: How does the unit definition for NEBExpress® Salt Active Nuclease compare to other similar salt-active nucleases? A: Although the unit definition assay conditions for NEBExpress Salt Active Nuclease differ (temperature, substrate, and buffer) from other similar nucleases on the market, we estimate that NEBExpress Salt Active Nuclease is up to 2 times more active compared to other salt nucleases. To switch from another salt nuclease to NEBExpress Salt Active Nuclease, we recommend testing a 1:1 substitution based on units. Q: Can I use NEBExpress® Salt Active Nuclease in a low salt buffer? A: Yes. NEBExpress Salt Active Nuclease retains 60% of its activity at 250 mM NaCl or KCl and 15% without salt. Depending on the application (viscosity reduction, partial or full nucleic acid degradation), enzyme concentration and incubation time may need to be increased at low salt. Q: Is NEBExpress® Salt Active Nuclease compatible with affinity columns such as IMAC? A: NEBExpress Salt Active Nuclease is not tagged and can be used to purify tagged proteins, such as a His-tagged protein by IMAC. Q: Can I remove NEBExpress® Salt Active Nuclease from my sample? A: Yes. NEBExpress Salt Active Nuclease will bind strongly to a cation exchange column due to its high pI, and, therefore, can be removed easily by capture on a cation exchange resin (Source 30S, SP HP, POROS 50HS resins are recommended). Q: After use, how can I inactivate NEBExpress® Salt Active Nuclease? A: NEBExpress Salt Active Nuclease can be inactivated by one of the following methods: Heat inactivation: Incubate at 65C for 2 h, at 80C for 1 h, or at 95C for 20 min. Chemical inactivation: Add > 10 mM EDTA, or 0.5% SDS, or > 20 mM TCEP. Chemical + heat inactivation: Add 1 mM DTT and incubate at 55C for 30 min. Enzymatic degradation: Add Proteinase K (NEB #P8107S) or Thermolabile Proteinase K (NEB #P8111S). Q: How are NEBExpress® Salt Active Nuclease and DNase I-XT (NEB #M0570) different? A: NEBExpress Salt Active Nuclease degrades DNA and RNA, while DNase I-XT degrades DNA only. It is active between 0 and 1000 mM salt concentration (optimal at 500 mM), while DNase-I XT has been engineered to be active between 0 and 300 mM salt. Furthermore, NEBExpress Salt Active Nuclease can be removed easily in a single column as it binds very efficiently to cation exchange resin. Q: What is the molecular weight of NEBExpress® Salt Active Nuclease? A: NEBExpress Salt Active Nuclease has a molecular weight of approximately 25 kDa. Q: Is NEBExpress® Salt Active Nuclease glycosylated? A: No. NEBExpress Salt Active Nuclease is produced from E. coli and, therefore, is not glycosylated, presenting as a single band at about 25 kDa on a protein gel.