Combination of 2DE and LC for Plant Proteomics Analysis - Plant Proteomics: Methods and Protocols - page 125

1 mM aprotinine), 4 % Triton X-100, and dithiothreitol
(DTT) 1 M.
2. Strips: Immobiline dry strips 24 cm, with a nonlinear pH
gradient 3-10 (GE Healthcare).
3. Rehydration buffer: 7 M urea, 2 M thiourea, 4 % CHAPS,
50 mM Tris-HCl, pH 8.0, 0.5 % IPG buffer pH 3-10 (GE
Healthcare), and 2 % DeStreak (GE Healthcare).
4. Equilibration buffer 1: 50 mM Tris-HCl, pH 8.8, 6 M urea,
30 % glycerol, 2 % SDS, 10 mg/mL DTT.
5. Equilibration buffer 2: 50 mM Tris-HCl, pH 8.8, 6 M urea,
30 % glycerol, 2 % SDS, 25 mg/mL iodoacetamide.
6. Staining solution: 0.1 % CBB R-250 (Bio-Rad), 10 % acetic
acid, 40 % methanol as previously described [ 9 ].
7. Lysis buffer 2: 6 M Urea, 2 M thiourea, 10 % glycerol, 50 mM
Tris-HCl, pH 8.0, 2 % (w/v) octyl- - D -glucopyranoside,

supplemented with 53 u/mL DNase I, 4.9 u/mL RNase, and
a cocktail of protease inhibitors (1 mM PMSF, 0.1 mM pep-
statin, 2 mM leupeptine, 1 mM E-64, and 1 mM aprotinine).
8. ProteomeLab PF-2D kit (Beckman Coulter): Contains start
buffer and elution buffer.
9. ProteomeLab PF-2D buffers for the second dimension:
Gradient with solvent A (0.1 % trifl uoroacetic acid (TFA)-
water) and solvent B (0.08 % TFA-acetonitrile) and wash
buffer (1 M NaCl).
10. Protein quantifi cation: BCA protein assay reagent Kit (Pierce).

β

3 Methods

1. Mature wheat dry embryos were manually dissected, collected,
frozen in liquid nitrogen, and stored at −80 °C until further
analysis [ 10 ].
2. Proteins from approximately 150 mg of embryos were
extracted with 1.2 mL lysis buffer 1 [ 11 ].
3. After 20-min incubation at 4 °C, 14 mM DTT was added and
samples were centrifuged for 10 min at 35,000 × g at 4 °C.
4. The supernatant was centrifuged once more and protein
content was quantifi ed using the BCA protein assay reagent
Kit (Pierce).

3.1 Protein
Extraction
and Quantifi cation
for 2DE Analysis

3.2 Protein
Extraction
and Quantifi cation
for LC Analysis

1. For the ProteomeLab PF-2D, proteins from approximately
150 mg of embryos were extracted with 1.2 mL of lysis buffer
2 [ 12 ] ( see Note 1 ).
2. Samples were centrifuged for 10 min at 35,000 × g at 4 °C.
The supernatant underwent a second centrifugation and