TurboNuclease can be used to
reduce viscosity of cell lysate and reduce back pressure of column loading
remove nucleic acid contamination from sample preparations, reduce nucleic acid contamination of Ni column purification
reduce smearing in SDS-PAGE when used with 10%SDS or Gel Loading Dye to make whole cell lysate
reduce or prevent clumping of concentrated cells and thawed cells
replaces crude DNase I in many applications
To reduce viscosity of cell lysate, 10-500 units of TurboNuclease can be used for each gram of cell paste. Generally, adding TurboNuclease to cell lysate at 25 U/ml is sufficient to reduce lysate viscosity.
The efficiency of viscosity reduction may vary with buffers, cell types, and cell lysis methods used. Due to its high specific activity, the total amount TurboNuclease added is less than 0.1 ug/ml of lysate and will not complicate any down stream process.
Lare Scale Cell Lysis
- Make fresh cold Lysis Buffer
Lysis Buffer should be a buffer in which the target protein is soluble.
The Lysis Buffer should be compatible with downstream purification processes, e.g. minimal amount of EDTA or DTT if Ni column will be used.
Here is an example of Lysis Buffer
25 mM Tris-HCl, pH 8.0
500 mM NaCl
14 mM beta-mercaptoethanol
Detergent can be included for less soluble proteins or when protein solubility is unknown. 1% Triton X-100 has no effect on TurboNuclease activity.
TurboNuclease has the same activity in 150 mM NaCl or 500 mM NaCl and 400 mM imidazole.
- Resuspend thawed cell paste in Lysis Buffer
Use 2-10 ml Lysis Buffer for each gram of cell paste. TurboNuclease can reduce the amount of Lysis Buffer used.
We routinely use 2 ml of lysis buffer for each gram of cell pellets. - Add TurboNuclease to 25 unit/ml
Protease inhibitors can be added at the same time.
If the lysis buffer contains EDTA or EGTA, add 10-fold more TurboNuclease. - Lyze cells by mechanical or chemical methods on ice or at room temperature
TurboNuclease also reduces the viscosity of lysate lyzed by microfluidizer. - Clear lysate by centrifugation for column loading
The reduced viscosity makes it possible to clear the lysate at lower speed. 35,000g (~16,000 rpm) for 1 hour is sufficient.
Lysate can be loaded to "Crude" columns without clearance.
Parallel Lysis of Multiple Insect Cell Samples
- Freeze cells pellets of 5-10 ml culture on dry ice briefly
Freeze and thaw facilitates lysis. - Thaw the frozen pellets and completely resuspend in ~1 ml Lysis Buffer with TurboNuclease
- Transfer the cell suspension to a microtube and sit the tubes on a floater rack
- Lyze cells using an Ultrasonic Cleaner with ice waterbath for 10 min
Ultrasonic Cleaner (many chemists use it) is much cheaper than probe sonicator. It costs a few hundreds US dollars for a new model and less than $100 for a used one or a jewelry cleaner from a consumer goods store. Ultrasonic Cleaner is much better and cheaper than those fancy multi-probe sonicators with the following advantages.
There is no cross-contamination since each sample is enclosed in a microtube.
The samples are always cold as long as ice is added in the water-bath.
There is no limit on the number of samples processed in parallel. A small Ultrasonic Cleaner can easily hold 48 samples
The lysate can be used for analyses of protein expression of whole cell lysate, soluble lysate, or affinity pull-down.
Eton Bio Lysis Buffer
25 mM Tris-HCl, pH 8.0
500 mM NaCl
20 mM Imidazole, pH 8.0
14 mM beta-mercaptoethanol
0.5% Triton X-100
25 units/ml TurboNuclease
Material Safety Data Sheet
Date:Oct 26,2015
1. PRODUCT AND COMPANY IDENTIFICATION
1.1 Product identifier
Product name: TurboNuclease
Catalog No:1400010050,1400010100,1400010250
1.2 Relevant identified uses of the substance or mixture and uses advised against
For research use only.
1.3 Details of the supplier of the safety data sheet
Company:Eton Bioscience Inc
TollFree:1-800-758-1630
Tel:1-800-758-1630
Fax:1-800-507-2912
1.4 Emergency telephone number
1-800-758-1630
2. HAZARDS IDENTIFICATION
2.1 Classification of the substance or mixture
Not a hazardous substance or mixture
2.2 GHS Label elements, including precautionary statements
Not a hazardous substance or mixture
2.3 Other hazards
None
3. COMPOSITION/INFORMATION ON INGREDIENTS
3.1 Substances
Synonyms : Recombinant Serratiamarcescensextracellular endonuclease
| CAS-No | EC-No. | Index-No | Classification | Concentration |
| Water |
| 7732-18-5 | 231-791-2 | - | - | >= 45 % |
| Glycerol |
|
|
|
|
| 56-81-5 | 200-289-5 | - | - | 50 % |
| Tris (hydroxymethyl) aminomethane free base |
| 77-86-1 | 201-064-4 | - | Xi, R36/37/38 | 0.6 % |
| Enzyme |
| - | - | - | - | <= 0.025 % |
| Magnesium chloride hexahydrate |
| 7791-18-6 | 232-094-6 | - | - | 0.1 % |
| Sodium chloride |
| 7647-14-5 | 231-598-3 | - | - | 0.3 % |
For the full text of the R-phrases mentioned in this Section, see Section 16.
4. FIRST AID MEASURES
4.1 Description of first aid measures
Eye contact
Check for and remove contact lenses and flush with copious amounts of water; assure adequate flushing by separating the eyelids with fingers; call a physician
Skin Contact
Flush with copious amounts of water; remove contaminated clothing and shoes; call a physician
Inhalation
Remove to fresh air. If not breathing give artificial respiration
Ingestion
If swallowed, never give anything by mouth to an unconscious person. Rinse mouth with water.
4.2 Most important symptoms and effects, both acute and delayed
No information available
4.3 Indication of any immediate medical attention and special treatment needed.
No information available
5. FIRE FIGHTING MEASURES
5.1 Extinguishing media
Suitable extinguishing media
Water spray, alcohol-resistant foam, dry chemical or carbon dioxide
5.2 Special hazards arising from the substance or mixture
No information available
5.3 Advice for firefighters
Wear self-contained breathing apparatus and protective clothing to prevent contact with skin and eyes.
6. ACCIDENTAL RELEASE MEASURES
6.1 Personal precautions,protective equipment and emergency procedures
Use full personal protective equipment. Evacuate personnel to safe areas. Avoid breathing vapors,mist or gas.
6.2 Environmental precautions
Prevent further leakage or spillage. Prevent product from entering drain.
6.3 Methods and material for containment and cleaning up
Keep in suitable, closed containers for disposal
7. HANDLING AND STORAGE
7.1 Precautions for safe handling
Avoid skin/eye contact. Use protective equipment as needed. Wash contaminated clothing before reuse.
7.2 Conditions for safe storage,including any incompatibilities
Keep container tightly closed. Store at -80?C.
7.3 Specific end use(s)
No data available.
8. EXPOSURE CONTROLS/PERSONAL INFORMATION
8.1 Control Parameters
This product contains no hazardous materials with occupational exposure limits.
8.2 Exposure controls
Engineering controls
Provide shower and eye wash station
Personal protective equipment
| Eye Protection | Wear safety goggles |
| Skin Protection | Wear protective clothing |
| Hand Protection | Wear protective gloves |
| Respiratory protection | Wear NIOSH/MSHA approved respirators |
9. PHYSICAL AND CHEMICAL PROPERTIES
9.1 Information on basic physical and chemical properties
| Appearance | Clear, colorless liquid |
| pH | 8.0 |
| Melting point | No data available |
| Flash point | No data available |
| Boiling point | No data available |
| Ignition temperature | No data available |
| Lower explosion limit | No data available |
| Upper explosion limit | No data available |
| Water solubility | Soluble |
9.2 Other safety information
No data available.
10. STABILITY AND REACTIVITY
10.1Reactivity
No data available
10.2Chemical stability
Stable under recommended storage conditions.
10.3Possibility of hazardous reactions
No data available.
10.4Conditions to avoid
No data available
10.5Incompatible materials
Strong oxidizing agents
10.6Hazardous decomposition products
Hazardous decomposition products formed under fire conditions: carbon oxides
11. TOXICOLOGICAL INFORMATION
11.1Information on toxicological effects
Acute toxicity
No data available
Irritation and corrosion
No data available
Sensitisation
Prolonged or repeated exposure may cause allergic reactions in certain sensitive individuals
Chronic exposure
IARC: No component of this product present at levels greater than or equal to 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.
Signs and Symptoms o Exposure
Prolonged or repeated exposure can cause:Nausea,Headache,Vomiting
Potential Health Effects
| Inhalation | May be harmful if inhaled. May cause respiratory tract irritation |
| Skin | May be harmful if absorbed through skin. May cause skin irritation |
| Eyes | May cause eye irritation |
| Ingestion | May be harmful if swallowed. |
| Target organs | Kidney |
12. ECOLOGICAL INFORMATION
12.1Toxicity
Avoid release into environment
12.2Persistence and degradability
No data available
12.3.Bioaccumulative potential
No data available
12.4Mobility in soil
No data available
12.5Results of PBT and vPvB assessment
No data available
12.6Other adverse effects
