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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Western blot analysis

Materials

TBS buffer

20 mM Tris-HCl, pH 7.5
150 mM NaCl

 

TBS-T buffer

20 mM Tris-HCl, pH 7.5
150 mM NaCl
0.05% Tween 20

 

 

Sample buffer

1%(w/v) Triton X
4 M Urea
50 mM Tris-HCl, pH 6.8
150 mM NaCl
2% (w/v) SDS
10% (w/v) glycerol
2% (w/v) mercaptoethanol

 

Blocking Solution

5% nonfat dry miik (included in detection kit, Bio-Rad, Cat.#170-6404) in TBS-T
TBS-T buffer

Precast Gels (7.5% Ready Gel Precast Gels, Bio-Rad, Cat.# 161-1118)

Mini Trans-Blot cell (Bio-Rad, Cat.# 170-3930)

Immun-Blot PVDF (Bio-Rad, Cat.# 162-0174)

Immun-Star^TM Goat Anti-Mouse HRP Detection Kit (Bio-Rad, Cat.#170-5044)

Primary Antibody

Anti-Renilla Luciferase (clone 5B11.2, Chemicon International, )
The dilution for primary antibody is1:2,000 from a stock solution

Secondary Antibody

Immun-Star^TM Goat Anti-Mouse-HRP Conjugate (Bio-Rad, Cat.#170-5047)
The dilution for the secondary antibody is 1:30,000 dilution

Protocols

1. Collect protoplast suspensions from 4 wells in a 96-well plate (a total of ~ 2x10⁴ protoplasts) into a 1.5 ml tube
2. Centrifuge at 100xg for 5 min.
3. Wash the protoplast pellets with TBS buffer.
4. Suspend the pellets in the sample buffer
5. Incubate at 95 °C for 5 min.
6. The lysates are loaded on a Precast Gel (7.5% Tris-HCl gel).
7. An electrophoresis is conducted at 200 Volts constant for 30 min.
8. An electrophoretic blotting at 100 Volts constant for 90 min is carried out with Mini Trans-Blot cell and Immun-Blot PVDF Membrane.
9. Signal detection is carried out with Immun-Star^TM HRP Chemiluminescent Kit.
   Blocking; Block with blocking solution for 60min at room temperature.
   Primary antibody incubation; Incubate the membrane with primary antibody for 60 min at room temperature with gentle agitation.
   Washing; Wash with 20 ml of TTBS for 3 times. Each wash is for 3 min with strong agitation at room temperature.
   Secondary antibody incubation; incubate the membrane secondary antibody for 60min at room temperature with gentle agitation.
   Washing; Wash with 20 ml of TTBS for 6 times. Each wash is for 3 min with strong agitation at room temperature.
   Blot development; Mix Luminol/enhancer and peroxide buffer solution in a 1:1 ration (6 ml each). Incubate the membrane in the substrate mixture for 3 min.
   Film exposure; Expose to X-ray file to visualize the band.

 

Last Update 8/1/07