TurboNuclease^TM

TurboNuclease^TM 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

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

1. 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.

2. 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.

3. 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.

4. Lyze cells by mechanical or chemical methods on ice or at room temperature
   TurboNuclease also reduces the viscosity of lysate lyzed by microfluidizer.

5. 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.

1. Freeze cells pellets of 5-10 ml culture on dry ice briefly
   Freeze and thaw facilitates lysis.

2. Thaw the frozen pellets and completely resuspend in ~1 ml Lysis Buffer with TurboNuclease

3. Transfer the cell suspension to a microtube and sit the tubes on a floater rack
   

4. 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.

   Accelagen 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