- How active should I expect it to be at 700 mM NaCl?
- Will SAN work in Triton X-100?
- My buffer also contains 10% glycerol… is this a problem?
- How much SAN would you add to 100 mL of lysate? I am pre-treating with polyethyleneimine and then want to use SAN to absolutely eliminate remaining DNA (essential for my enzyme purification).
- Can I replace Benzonase with SAN in all applications?
- What is the advantage of salt-active SAN over non salt-active DNases?
- What might be the disadvantage of the salt-active properties of SAN?
- How to use SAN for reduction of viscosity in cell samples?
- What is the inactivation temperature of SAN?
- How to inactiavte/remove SAN after use?
- What is the activity of SAN in protein extraction buffers?
- What is the lowest reaction temperature I can use for SAN?
- What salt concentration is optimal for SAN?
- What pH is optimal for SAN?
- What is the recommended amount of SAN per reaction?
- What is the specific activity of SAN?
- Is SAN a tagged protein?
- Does the SAN preparation contain BSA?
- How stable is SAN?
- What is the difference between SAN and HL-SAN?
- How can I visually check whether my SAN is really active and working?
The activity at 700 mM NaCl will be high – it is very near the optimal salt concentration.
Yes. No loss of activity is detected in presence of up to 15% Triton X-100
Glycerol will inhibit the activity and at 10% glycerol SAN will have ~80% activity compared to a buffer containing no glycerol. There is little or no activity left at 50% glycerol.
How much SAN would you add to 100 mL of lysate? I am pre-treating with polyethyleneimine and then want to use SAN to absolutely eliminate remaining DNA (essential for my enzyme purification).
This depends on several factors, as strain, buffer, detergents, salt concentration, temperature etc. Much more SAN is needed for total DNA removal compared to what is adequate for viscosity reduction. In a typical bacterial lysate containing 0.5 M NaCl we recommend 1 kU pr ml of lysate if you want to incubate at room temperature, but less can be used if incubation temperature is increased. It is also possible to incubate at 4°C, but then more SAN or increased incubation time is needed. In case most of the nucleic acids is removed in a pre-treatment using polyethyleneimine less SAN than normal recommendation can be used.
Probably not – SAN has a different activity profile and tolerance for some buffer components. But in cases where you need a non-specific nuclease that works in high-salt buffers and is easier to remove after use, SAN is superior.
In order to release proteins from DNA, salt is often added to protein extraction buffers. This makes DNA accessible for degradation, but most DNases are not very active at such high-salt conditions. SAN has its optimum activity at high salt conditions, upon which DNA is accessible for degradation.
As salt is not only tolerated, but also required for optimal activity of SAN – the enzyme might not be as efficient as required in situations where the buffers contain very low amounts of salt.
Add SAN to your cell sample, about 25 U per ml of lysate containing 0.5 ml NaCl and incubate at room temperature.
SAN is not easy to inactivate by heating alone. Half life at 70°C is about 2 hours. However, SAN is susceptible to reducing agents, and addition of e.g. DTT or TCEP enhances the inactivation of the enzyme. See, “How to inactivate/remove SAN after use”, below.
There are several strategies one can use to inactivate or remove SAN after use. SAN is susceptible to reducing agents such as DTT or TCEP even at moderate temperatures (25-55°C). A concentration of reducing agent of 1-10 mM is recommended. Inactivation of SAN will depend on the concentration of the reducing agent, inactivation time and temperature. Addition of chelators such as EDTA will inhibit the SAN activity by binding Mg2+. The pI of SAN is very high (9.6), which means that it in many cases can be removed using ion exchange chromatography.
The activity of SAN in protein extraction buffers depends on the buffer composition, and has to be determined experimentally. Detergents, which are often constituents of lysis buffers, will inhibit the activity of SAN to some degree, and Mg2+ is required for activity.
The optimal reaction temperature of SAN is 37°C. However, SAN is active at low temperatures also, but the reaction will be slower. Activity at 4°C is about 5-10% of activity at 37°C. You may compensate by increasing the reaction time or increasing the amount of enzyme.
0.5 M NaCl is optimal in a reaction buffer of pH 8.5. However, low concentration of salt can be compensated for by using higher pH.
pH 9. If using a lower pH, this can compensated for by using a higher concentration of salt (0.5-1.0 M NaCl).
This strongly depends on the reaction conditions. At recommended conditions (pH 8.5, 5 mM MgCl2, 0.5 M NaCl), ≤10 U SAN per mL (0.1 per μL) reaction completely removes 0.5 μg/μL DNA during a 30-min incubation at 37°C.
In typical bacterial lysates, including 0.25-1.0 M NaCl, 1000 U SAN per mL lysate is needed to completely remove DNA (incubation for 30 min at r.t.). If total removal of DNA is important, it is advantageous to incubate the lysate overnight in 0.5 M NaCl at 4°C.
Please contact firstname.lastname@example.org if specific recommendations are needed.
About 500,000 U/mg
SAN is a robust enzyme which is stable at room temperature for weeks. We recommend storage at -20°C or 4°C for best storage stability.
HL-SAN is more sensitive to reducing agents than SAN, which makes it possible to completely inactivate HL-SAN using the right combination of reducing agent, inactivation time and temperature.
SAN will degrade DNA and RNA, and a cell lysate will be visually less viscous. Any method for nucleic acid detection can be used to check for remaining nucleic acids. Agarose gel electrophoresis is an easy way to test this.