4. Glass beaker.
5. Parafilm.
6. Weighing balance.
2.2 Apoplastic Fluid
Isolation Buffers
and Equipment
1. Ice-cold deionized water: Store several 1-L glass bottles at
4
C.
2. Coffee infiltration buffer: 100 mM Tris–HCl buffer, pH 7.6;
add about 100 mL of water to a 1-L graduated a glass beaker,
weigh 121.14 g of Tris and transfer to the beaker, add water to
a volume of 900 mL, and mix with a magnetic stir bar till it
dissolves. Add 500 mM L-ascorbic acid (88.06 g) and 500 mM
potassium chloride (37.28 g) and mix till it dissolves (see Note
3). Adjust pH with HCl. Make up to 1 L with water. Keep it at
4
C for no longer than 2 weeks. Add 25 mM
2-mercaptoethanol (1.7 mL per 100 mL of buffer) to the
buffer solution just before use (see Note 4) [8, 9].
3. Grapevine infiltration buffer: 100 mM Tris–HCl buffer,
pH 7.6; add about 100 mL of water to a 1-L graduated a
glass beaker, weigh 121.14 g of Tris and transfer to the beaker,
add water to a volume of 900 mL, and mix with a magnetic stir
bar till it dissolves. Adjust pH with HCl. Add 500 mM potassium chloride (37.275 g) and 6 mM CHAPS (3.68 g) (see Note
5) and mix till it dissolves. Make up to 1 L with water. Keep it at
4
C.
Add 2% of sodium sulfite (20 g/L) to the buffer solution
just before use (see Note 6) [10, 11].
4. Soft towel paper.
5. Large glass Kitasato flask, or vacuum flask, with lid.
6. Vacuum pump to apply vacuum at 25 kPa (187.52 mmHg).
7. 20-mL plastic syringe without a plunger.
8. 50-mL falcon tube or equivalent.
9. 1.5-mL Eppendorf tubes.
10. Refrigerated centrifuge with fixed angle rotor for 50-mL centrifuge tubes.
2.3 Apoplast Protein
Concentration
and Quantification
1. Refrigerated centrifuge with fixed angle rotor for 15 mL tubes.
2. Centrifugal filter Vivaspin2.
3. Eppendorf tubes (1.5 mL).
4. Micropipettes.
5. Bradford reagent.
6. Protein standard: 2.5 mg/mL albumin in water.
7. 96 ELISA well microplates.
8. ELISA reader (ABS 595 nm).
Extraction of Apoplastic Fluid from Woody Plants
51
5. Parafilm.
6. Weighing balance.
2.2 Apoplastic Fluid
Isolation Buffers
and Equipment
1. Ice-cold deionized water: Store several 1-L glass bottles at
4
C.
2. Coffee infiltration buffer: 100 mM Tris–HCl buffer, pH 7.6;
add about 100 mL of water to a 1-L graduated a glass beaker,
weigh 121.14 g of Tris and transfer to the beaker, add water to
a volume of 900 mL, and mix with a magnetic stir bar till it
dissolves. Add 500 mM L-ascorbic acid (88.06 g) and 500 mM
potassium chloride (37.28 g) and mix till it dissolves (see Note
3). Adjust pH with HCl. Make up to 1 L with water. Keep it at
4
C for no longer than 2 weeks. Add 25 mM
2-mercaptoethanol (1.7 mL per 100 mL of buffer) to the
buffer solution just before use (see Note 4) [8, 9].
3. Grapevine infiltration buffer: 100 mM Tris–HCl buffer,
pH 7.6; add about 100 mL of water to a 1-L graduated a
glass beaker, weigh 121.14 g of Tris and transfer to the beaker,
add water to a volume of 900 mL, and mix with a magnetic stir
bar till it dissolves. Adjust pH with HCl. Add 500 mM potassium chloride (37.275 g) and 6 mM CHAPS (3.68 g) (see Note
5) and mix till it dissolves. Make up to 1 L with water. Keep it at
4
C.
Add 2% of sodium sulfite (20 g/L) to the buffer solution
just before use (see Note 6) [10, 11].
4. Soft towel paper.
5. Large glass Kitasato flask, or vacuum flask, with lid.
6. Vacuum pump to apply vacuum at 25 kPa (187.52 mmHg).
7. 20-mL plastic syringe without a plunger.
8. 50-mL falcon tube or equivalent.
9. 1.5-mL Eppendorf tubes.
10. Refrigerated centrifuge with fixed angle rotor for 50-mL centrifuge tubes.
2.3 Apoplast Protein
Concentration
and Quantification
1. Refrigerated centrifuge with fixed angle rotor for 15 mL tubes.
2. Centrifugal filter Vivaspin2.
3. Eppendorf tubes (1.5 mL).
4. Micropipettes.
5. Bradford reagent.
6. Protein standard: 2.5 mg/mL albumin in water.
7. 96 ELISA well microplates.
8. ELISA reader (ABS 595 nm).
Extraction of Apoplastic Fluid from Woody Plants
51