1
Grind sample to fine
power in liquid nitrogen
Homogenize in extraction buffer (lysis buffer)
[7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer
pH 3-10 NL, 65 mM DTT] + protease inhibitor mix
Centrifuge at 15000xg, 30 min, 20 °C
x2
Transfer the supernatant to new tube
Centrifuge at 15000xg, 20 min, 20 °C
Transfer the supernatant to new tube
Fig. i A flow chart of direct lysis buffer extraction method used in this study.
2
A
B
Grind sample to fine
power in liquid nitrogen
Grind sample to fine
power in liquid nitrogen
Add 1% PVPP
Re-suspend in TCA/acetone solution
[10% TCA, 0.07% 2-ME]
Homogenize in extraction buffer
[20 mM Tris-HCl pH 8.0, 2% NP-40,
1 mM EDTA, 1 mM PMSF]
Incubate at -18 °C, 45 min
Centrifuge at 35000xg, 15 min
Centrifuge at 23000xg, 30 min, 4 °C
Wash pellet with acetone solution
[0.07% 2-ME in acetone]
Transfer the supernatant to new tube
Incubate at -18 °C, 1 h
Centrifuge again
Centrifuge at 23000xg, 30 min, 4 °C
Dry pellet under vacuum
Re-suspend in UKS solution
[9.5 M urea, 5 mM K2CO3, 6% Triton X-100,
1.25% SDS, 2% LKB ampholines, 0.5% DTT]
Precipitate with 5 vol. TCA/acetone solution
[12% TCA, 24 mM DTT]
Final conc. of 10% TCA
Incubate at -20 °C overnight
Centrifuge at 23000xg, 30 min, 4 °C
Centrifuge 14000xg, 5 min
Wash pellet with acetone solution
[20 mM DTT in acetone]
x3
Incubate at -20 °C, 10 min
Centrifuge at 23000xg, 5 min, 4 °C
Air dry and re-suspend in lysis buffer
[7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG
buffer pH 3-10 NL, 65 mM DTT]
Fig. ii The flow charts of TCA/acetone precipitation method for protein preparation with a classical protocol
developed by Damerval et al. (1986) (A) and the modified method used in this study (B).
3
A
B
Prepare plasma
membrane sample
Grind sample to fine
power in liquid nitrogen
Add 1% PVPP
Homogenize in extraction buffer
[0.5 M Tris, 30 mM HCl, 0.7 M sucrose, 50 mM
EDTA, 2 mM PMSF, 0.1 M KCl, 2% 2-ME]
Homogenize in extraction buffer
[0.5 M Tris-HCl pH 8.0, 0.7 M sucrose, 5 mM
EDTA, 1 mM PMSF, 0.1 M KCl, 20 mM DTT]
Incubate at 4 °C, 10 min
Vortex 5 min, incubate on ice, 10 min
Add equal volume of
water-saturated phenol
Add an equal volume of Tris-saturated
phenol pH 8.0 (with trace bromophenol blue)
Shake at room temperature, 10 min
Centrifuge
Vortex 5 min, incubate on ice, 10 min
Centrifuge at 15000xg, 15 min, 20 °C
Transfer phenol phase to new tube
Transfer phenol phase to new tube
15000xg,
10 min, 20 °C
Re-extract with an equal
volume of extraction buffer
x2
Re-extract with an equal
volume of extraction buffer
Precipitate with 5 vol. precipitation solution
[0.1 M ammonium acetate in methanol]
Precipitate with 5 vol. precipitation solution
[0.1 M ammonium acetate, 20 mM DTT
in methanol]
Incubate at -20 °C overnight
Incubate at -20 °C overnight
Centrifuge at 15000xg, 30 min,20 °C
Wash pellet with precipitation solution
Wash pellet with precipitation solution
x3
x3
Incubate at -20 °C, 30 min
Centrifuge at 15000xg, 10 min, 20 °C
Wash pellet with wash solution
[20 mM DTT in 90% ethanol]
Wash pellet with acetone
x2
Dry under nitrogen gas stream
Re-suspend in solubilization buffer
[9 M urea, 4% NP-40, 2% ampholytes,
2% 2-ME]
Incubate at -20 °C, 30 min
Centrifuge at 15000xg, 10 min, 20 °C
Air dry and re-suspend in lysis buffer
[7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG
buffer pH 3-10 NL, 65 mM DTT]
Fig. iii The flow charts of phenol extraction method for protein preparation with a classical protocol developed
by Hurkman and Tanaka. (1986) (A) and the modified method used in this study (B).
4
B
A
Grind sample to fine
power in liquid nitrogen
Grind sample to fine
power in liquid nitrogen
Add 1% PVPP
Homogenize in extraction buffer
[0.5 M Tris-HCl pH 8.3, 2% NP-40, 1 mM PMSF,
20 mM MgCl2, 2% 2-ME, 1% PVPP]
Homogenize in extraction buffer
[0.5 M Tris-HCl pH 8.0, 2% NP-40, 1 mM EDTA,
1 mM PMSF, 20 mM MgCl2, 40 mM DTT]
Centrifuge at 12000xg,15 min, 4 °C
Vortex 5 min, incubate on ice, 10 min
Centrifuge at 15000xg,15 min, 20 °C
Transfer the supernatant to new tube
Transfer the supernatant to new tube
Centrifuge at 15000xg, 30 min, 20 °C
Transfer the supernatant to new tube
Add 50% PEG solution
Add 3/7 vol. of 50% PEG solution
Final conc. of 10% PEG
Final conc. of 15% PEG
Incubate on ice, 30 min
Centrifuge at 1500xg,10 min
Vortex 1 min, incubate on ice, 30 min
Centrifuge at 13000xg,15 min, 20 °C
Re-suspend PF with
extraction buffer
PF (10%)
Centrifuge at 15000xg,
15 min, 20 °C
Transfer the supernatant to new tube
Transfer the supernatant to new tube
Add 50% PEG solution
SF
Final conc. of 20% PEG
Incubate on ice, 30 min
Centrifuge at 12000xg, 15 min
PF
Precipitate with 5 vol. precipitation
solution [20 mM DTT in acetone]
PF (20%)
Incubate at -20 °C overnight
Centrifuge at 15000xg, 15 min, 20 °C
Transfer the supernatant
to new tube
Wash pellet with precipitation solution
Precipitate pellet
with acetone
SF
x3
x3
Incubate at -20 °C, 10 min
Centrifuge at 15000xg, 10 min, 20 °C
Re-suspend in solubilization
buffer [9.5 M urea, 2% NP-40,
2% ampholines, 5% 2-ME]
Air dry and re-suspend in lysis buffer
[7 M urea, 2 M thiourea, 4% CHAPS,
2% IPG buffer pH 3-10 NL, 65 mM DTT]
Fig. iv The flow charts of PEG fractionation method for protein preparation with an original protocol
developed by Kim et al. (2001) (A) and the modified method used in this study (B).
5
A
Fig. v The synthetic gels of 2-DE protein profiles from rice seeds (A) and seedlings (B) extracted using direct lysis buffer extraction method.
All spots are the consistent protein spots and the opaque green spots represent the unique spots.
B
6
A
Fig. vi The synthetic gels of 2-DE protein profiles from rice seeds (A) and seedlings (B) extracted using TCA/acetone precipitation method .
All spots are the consistent protein spots and the opaque green spots represent the unique spots.
B
7
A
Fig. vii The synthetic gels of 2-DE protein profiles from rice seeds (A) and seedlings (B) extracted using phenol extraction method .
All spots are the consistent protein spots and the opaque green spots represent the unique spots.
B
8
A
Fig. viii The synthetic gels of 2-DE protein profiles from rice seeds extracted using PEG fractionation method that include pellet (A) and supernatant fraction (B).
All spots are the consistent protein spots and the opaque green spots represent the unique spots.
B
9
A
Fig. ix The synthetic gels of 2-DE protein profiles from rice seedlings extracted using PEG fractionation method that include pellet (A) and supernatant fraction (B).
All spots are the consistent protein spots and the opaque green spots represent the unique spots.
B