[Efficient protein extraction method from apple leaves for apple proteomic analysis using two-dimensional electrophoresis analysis].
In order to develop an efficient protein extraction method suitable for apple leaf proteomic analysis, four extraction methods for total protein in apple leaves were compared, including trichloroacetic acid (TCA)/acetone precipitation, dithiothreitol (DTT)/acetone method, tri(hydroxymethyl) aminomethane (Tris-HCl) method and the modified Tris-HCl method. During the two-dimensional electrophoresis (2-DE), the first dimension electrophoresis was performed on a 7 cm strip with pH 3 - 10 linear immobilized pH gradient (IPG) and the second one was performed on 12.5% polyacrylamide gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were detected by silver staining. The results showed that 140, 215, 181 and 616 protein spots were detected on 2-DE gels, respectively. The modified Tris-HCl method was the most appropriate for apple leaf proteomic analysis because of the highest resolution and no apparent vertical or horizontal streaking on the 2-DE map. In order to testify the effect of the modified Tris-HCl method on the apple leaf protein extraction, 2-DE maps were established by using 18 cm strips with linear IPG in pH range of 3 - 10. After 2-DE separation and Coomassie Brilliant Blue R-250 (CBB R-250) staining, about 455 spots were detected, and the relative molecular masses of most proteins were distributed in the range of 14,000 - 66,000 which were free of smearing or streaking. So it was once again proved that the modified Tris-HCl method can be used in apple leaf proteome analysis.
Zeng
Guanjuan
G
College of Horticulture, Shenyang Agricultural University, Skenyang, China.
Li
Chunmin
C
Zhang
Xinzhong
X
Teng
Yunlong
Y
Dong
Wenxuan
W
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D027845:Malus / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry*; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification*
D010940:Plant Proteins; D020543:Proteome