Fig 1.
Comparison between the steps of the modified TCA/acetone precipitation, the classical TCA/acetone precipitation, and acetone precipitation methods.
The SDS extraction buffer contained 1% (w/v) SDS, 0.1 M Tris-HCl (pH 6.8), 2 mM EDTA-Na2, 20 mM DTT, and 2 mM PMSF (added before use). All organic solvents were pre-chilled at -20°Cand contained 5 mM DTT (added before use).
Fig 2.
Optimization of TCA/acetone ratio used in the modified method.
Equal amounts (ca. 30 μg) of maize root proteins were analyzed by SDS-PAGE (12.5% resolving gel). Protein was stained using CBB. A, final acetone concentration was 50% (v/v), but TCA concentration varied from 0–20% (w/v) in the aqueous mixture. B, final TCA concentration was 10% (w/v), but acetone concentration varied from 0–80% (v/v) in the aqueous mixture.
Table 1.
Comparison of protein yield and spot number in 2DE between the two methods.
Fig 3.
Comparison of 2DE protein profiles of maize embryo proteins extracted using two methods.
Left panel: the modified TCA/acetone precipitation. Right panel: the classical TCA/acetone precipitation. Spots with increased abundance are indicated in red. About 800 μg of proteins were resolved in pH 4–7 (linear) strip by IEF and then in 12.5% gel by SDS-PAGE. Proteins were visualized using CBB.
Table 2.
The identification of the differential extracted proteins in maize using the two methods.
Fig 4.
Comparison of 2DE profiles of maize mesocotyl proteins extracted using two methods.
Another two independent experiments were shown in S4 Fig. Left panel: the modified TCA/acetone precipitation. Right panel: acetone precipitation. About 800 μg of proteins were resolved in pH 4–7 (linear) strip by IEF and then in 12.5% gel by SDS-PAGE. Protein was visualized using colloidal CBB.