Overview of our system

Vector information

Protocols

Protoplast isolation

Gene transfection

Luciferase assay

Western blotting

Results

Transfect efficiency

Gene expression levels of dual expression vectors

Protein interaction

Acknowledgements

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Updated Protoplast preparations for gene transfection (11/30/09)

Protoplast isolation protocol is based on the method developed by Sheen and colleagues (Sheen, J. 2002, A transient expression assay using Arabidopsis mesophyll protoplasts.). The protocol establsihed for our lab can be downloaded from here.

Materials

• razor blade(single edge carbon steel, Electron Microscopy Sciences, Cat.#71950)
• 100 μm nylon mesh (cell strainer 100 μm, BD Biosciences, Cat.#352360)

Digestion buffer

1%(w/v) cellulase "onozuka"R10 (Karlan Research Products Corp. or Yakult Pharmaceutical industry co.,ltd.)
0.25%(w/v) macerozyme R10 (Karlan Research Products Corp. or Yakult Pharmaceutical industry co.,ltd)
0.4 M mannitol
20 mM MES (pH 5.7)
20 mM KCl
10 mM CaCl₂

Preparation

1. Add 100 mg of cellulase R10 and 25 mg of macerozyme R10 into a 50 ml tube.
2. Add 10 ml of buffer containing 0.4 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 10 mM CaCl₂ into the tube.
3. Vortex the tube briefly, and shake in orbit at room temperature for few min (2-5 min).
   *The digestion solution will be clear (light brown).
   **No heat treatment and No filtration

W5 buffer

154 mM NaCl
125 mM CaCl₂
5 mM KCl
2 mM MES (pH 5.7)

 

MMg solution

0.4 M mannitol
15 mM MgCl₂
4 mM MES (pH 5.7)

Protocols

1. About one-month old Arabidopsis leaves (0.5 - 1.0 g) are cut into 1-2 mm strips with a razor blade on a copy paper.
2. The leaf strips are transferred to 10 ml of digestion buffer in a Petri dish.
3. The dish is then vacuum for 30 min at room temperature to permeate the buffer into the stripped leaves.
4. The dish is placed in the dark for 6 hours at room temperature (25 °C).
5. 10 ml of W5 buffer are added to the Petri dish.
6. Filtere the protoplast suspension into a 50 ml tube with a 100 μm nylon mesh.
7. Collect the protoplasts by centrifugation at 100×g for 3 min.
8. Wash two times in 10 ml of W5 buffer through consecutive resuspension and centrifugation at 100×g for 3 min.
9. Resuspend the protoplasts in 10 ml of W5 solution and are kept at 4 °C for 30 min.
10. Collect the protoplasts by centrifugation at 100×g for 3 min.
11. Wash in 5 ml of MMg solution.
12. Resuspend the protoplast in 5 - 10 ml of the MMg solution to count the numbers in the suspension with a haematocytometer.

Pre-digestion	Post-digestion	Protoplast

 

 

Last Update 9/23/10