Biology Reference
In-Depth Information
6. Seedling pot (55 × 50 × 55 mm, T. O. Plastics, MN, USA).
7. Fourth or fi fth leaf stage of rice seedling (
see
Note 3
).
8. Liquid nitrogen.
9. Sterile/clean mortar and pestle.
10. 40 mL Nalgene centrifuge tubes (Rochester, NY, USA) and
1.5 mL microfuge tubes (Sarstedt, Nümbrecht, Germany).
1. Rice leaves (
O. sativa
L. cv. Jinheung) (4th and 5th leaf stages)
with/without
M. oryzae
or
X. oryzae
infection.
2. Phenol: Tris-HCl, pH 7.9, saturated (Amresco, Solon, OH,
USA).
3. Mg/NP-40 extraction buffer: 0.5 M Tris-HCl, pH 8.3, 2 %
v/v Nonidet P (NP)-40 (Amresco, Solon, OH, USA), and
20 mM MgCl
2
. This solution can be stored at 4 °C.
Just before extraction, add 2 % (v/v)
2.4 Preparation
of Total Protein
Extraction: Phenol
Extraction
-mercaptoethanol
(Amresco, Solon, OH, USA), 1 mM phenylmethylsulfonyl fl uoride
(PMSF; Merck, Whitehouse Station, NJ, USA), and 1 % w/v poly-
vinylpolypyrrolidone (PVPP; Sigma, St. Louis, MO, USA).
4. Washing solution: 80 % (v/v) acetone (SK Chemical, Ulsan,
South Korea) in deionized water. This solution can be stored
at −20 °C.
5. Precipitation solution: 100 % methanol (Burdick & Jackson,
Morristown, NJ, USA) containing 0.1 M ammonium acetate
(Sigma, St. Louis, MO, USA). This solution can be stored
at −20 °C.
β
2.5 Preparation of
Polyethylene Glycol
(PEG) Fractionation
1. Mg/NP-40 extraction buffer.
2. 50 % PEG 4000 solution (Sigma, St. Louis, MO, USA).
3. 100 % methanol containing 0.1 M ammonium acetate.
4. Tris-HCl, pH 7.9, saturated phenol.
2.6 In Planta
Secretome
1. Nalgene 250 mL centrifuge bottle.
2. Extraction buffer (CA buffer): 200 mM CaCl
2
(Sigma, St.
Louis, MO, USA), 5 mM Na-acetate (Sigma, St. Louis, MO,
USA), pH 4.3.
3. No.2 fi lter paper (Advantec MFS, Dublin, CA, USA).
4. Tris-HCl, pH 7.9, saturated phenol.
5. Precipitation solution: 100 % methanol containing 0.1 M
ammonium acetate.
6. Resuspension solution: 80 % methanol containing 0.1 M
ammonium acetate.
7. Washing solution: 80 % acetone.
