Team:Kyoto/Protocol

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Contents

Protocol

Western blotting

Gel solution
Running gelStacking gel
1.5M Tris-HCl(pH8.8)2.5mL-
0.25M Tris-HCl(pH6.8)-3.0mL
30% Acrylamide5mL0.6mL
10% SDS0.2mL0.12mL
DW2.3mL2.3mL
TEMED15µL7µL
10% APS100µL60µL
Total10mL6mL
4x Sample buffer
 1M Tris-HCl      2mL
 SDS              0.8g
 100% glycerol     4mL
 14.7M mercaptoethanol 0.4mL
 0.5M EDTA       1mL
 Bromophenol blue 8.0mg
 DW              2.6mL
10x electrode buffer
 Tris       15.15g
 Glycin     71.55g
 10%SDS   50mL
 DW       450mL
 Total      500mL
blotting buffer
 Tris       12g
 Glycin    14.4g
 DW       800mL
 Methanol  200mL
TBST
 50mM     Tris
 150mM    NaCl
 0.1%      Tween-20
blocking buffer
 TBST
 5%  skim milk
AP color development buffer
 100mM  Tris (pH8.5)
 100mM  NaCl
 5mM    MgCl2

1. Spin down 100µL culture and suspend into sample buffer.
2. Boil at 95°C for 10 min.
3. Apply 10µL to polyacrylamide gel.
4. Electrophorese at 500V, 30mA for 50 min.
5. Transfer at 50V, 100mA for 30 min.
6. Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.
7. Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.
8. Wash with TBST and incubate with shaking for 10 min, two times.
9. Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.
10. Wash with TBST and incubate with shaking for 10 min, three times.
11. Add 66µL NBT and 33µLof BCIP into color development buffer., and add the mixture to the membrane.
12.After the coloring, wash with DW.

Verification of R9 function by GFP

Verification of R9 function by TAKEUCHI

R9(20µg/µL)0.9µL
GFP(1.2mg/mL)2.23µL
RBS16.85µL
total20µL

X5

Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.

123456
R9oooxoo
cuticleooooxx
soak in GFP5min15min30min5min5min30min