Total protein extracts were prepared from dry mature seeds, and from seeds at different stages of germination. Following grinding of seeds using mortar and pestle (150 mg representing approximately 8 400 wild type seeds) in liquid nitrogen, total proteins were extracted at 2°C in 2.2 ml of thiourea/urea lysis buffer (Harder et al
., 1999) containing :
7 M urea (Amersham Pharmacia Biotech, Orsay, France),
2 M thiourea (Merck, Lyon, France),
4% (w/v) 3-[(3-cholamidopropryl)dimethylammonio]-1-propanesulfonate (CHAPS) (Amersham Pharmacia Biotech, Orsay, France),
1% (v/v) Pharmalyte pH 3-10 carrier ampholytes (Amersham Pharmacia Biotech, Orsay, France).
18 mM Tris-HCl (Trizma HCl; Sigma, St Quentin Fallavier, France),
14 mM Trizma base (Sigma, St Quentin Fallavier, France),
0.2% (v/v) Triton X-100 (Sigma, St Quentin Fallavier, France),
the protease inhibitor cocktail “complete Mini” from Roche Diagnostics GmbH (Mannheim, Germany),
53 U/ml DNase I (Roche Diagnostics GmbH, Mannheim, Germany),
4.9 Kunitz U/ml RNase A (Sigma, St Quentin Fallavier, France).
Protease inhibitor, DNase I and RNase A was added in the extraction buffer in a last step.
After 10 min at 4°C, 14 mM dithiothreitol (DTT) (Amersham Pharmacia Biotech, Orsay, France) was added and the protein extracts were stirred for 20 min at 4°C, then centrifuged (35000 g, 10 min) at 4°C. The supernatant was submitted to a second clarifying centrifugation as above. The final supernatant corresponded to the total protein extract. Protein concentrations in the various extracts were measured according to Bradford (1976). Bovine serum albumin was used as a standard.
- Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal Biochem 72, 248-254
- Harder A, Wildgruber R, Nawrocki A, Fey SJ, Larsen PM, Görg A (1999) Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Electrophoresis 20, 826-829