Protocol

Western blotting

Gel solution

	Running gel	Stacking gel
1.5M Tris-HCl(pH8.8)	2.5mL	-
0.25M Tris-HCl(pH6.8)	-	3.0mL
30% Acrylamide	5mL	0.6mL
10% SDS	0.2mL	0.12mL
DW	2.3mL	2.3mL
TEMED	15µL	7µL
10% APS	100µL	60µL
Total	10mL	6mL

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
RBS	16.85µL
total	20µL

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.

	1	2	3	4	5	6
R9	o	o	o	x	o	o
cuticle	o	o	o	o	x	x
soak in GFP	5min	15min	30min	5min	5min	30min

Team:Kyoto/Protocol

From 2012.igem.org

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