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.