The main advantages of SAN
- Removes RNA and DNA
- Very active at high salt concentration
- Active at low temperatures
- Stable at room temperature
To increase protein solubility in a cell lysate, high concentrations of salt are commonly added. The reason is that DNA-protein complexes usually dissociates with increasing salt-concentrations, making DNA more available for nuclease activity.
Salt Active Nuclease (SAN) is an excellent enzyme to remove DNA and RNA in buffers and cell extracts with moderate and high salt content. SAN degrades nucleic acids and thus reduces viscosity of cell lysates.
SAN has also been used in virus processing to reduce viral aggregation in high salt buffers.
At conditions where other nucleases are inhibited, SAN becomes more active!
DNase I and Benzonase are the most commonly used endonucleases. However in high salt conditions, these enzymes are not very efficient. SAN works better in higher salt and Mg2+ conditions.
|Capability (or feature)||SAN||Benzonase||DNase I|
|Salt tolerance, Na+||Optimal||500 mM||0-20 mM||0-20 mM|
|Effective||50 mM-1 M||0-150 mM|
|Mg2+||Optimal||5-10 mM||1-2 mM||5-10 mM|
|Effective||1-40 mM||1-10 mM|
|Reducing agents*||Tolerance||0 mM||0-100 mM|
|Table 1. Operating conditions for different products.
* Examples: DTT, TCEP, ß-Me.
|Figure 1. SAN activity compared to Benzonase and DNaseI.|
SAN has a much higher activity than Benzonase with higher salt concentration and lower temperature.
Activity in different buffer conditions
|Salt optimum: 0.5 M NaCl pH optimum: pH 9|
- Storage buffer: 25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.5 M NaCl, 0.01% Triton, 50% (v/v) glycerol.
- Reaction buffer: 25 mM Tris-HCl, pH 8.5 (25ºC), 5 mM MgCl2, 500 mM NaCl, 50 μg/ml calf thymus DNA.
- Unit definition: One Unit is defined as an increase in absorbance at 260 nm of 1 A in 30 minutes at 37ºC, using 50 μg/ml calf thymus DNA (D-1501, Sigma) in a buffer consisting of 25 mM Tris-HCl, pH 8.5 (25ºC), 5 mM MgCl2, 500 mM NaCl.
- Specific activity: 400.000 Units/mg.
- LitR Is a Repressor of syp Genes and Has a Temperature-Sensitive Regulatory Effect on Biofilm Formation and Colony Morphology in Vibrio(Aliivibrio) salmonicida
Appl. Environ. Microbiol. September 2014 vol. 80 no. 17 5530-5541
Published ahead of print 27 June 2014, doi: 10.1128/AEM.01239-14