Leaf Protein Extraction Methods Suitable for SDS-PAGE Analysis in Apple

In order to develop an efficient protein extraction method suitable for SDS-PAGE analysis,five protocols for protein extraction in apple leaf,trichloroacetic acid/acetone precipitation(TCA),modified Tris-HCl extraction,phenol extraction methanol/ammonium acetate precipitation,DTT/acetone and Tris-HCl extraction were compared.The extracts were separated by SDS-PAGE method followed by silver staining.The results showed that the modified Tris-HCl extraction presented the highest resolution of 3.243 μg/μL and the maximum bands of 41 strips with the 5 μL sample volumes basing on the increased amount of PVPP and the extracting solution as well as prolonged 30 min of protein precipitation with cold acetone.Dynamic changes of proteins in leaves from different nodes in apple seedlings(Jonathan×Golden Delicious)were analyzed with the improved Tris-HCl extraction method,six protein bands were detected with the molecular weight 71.9,60.5,52.6,41.1,35.3,18.5 kDa respectively and the bands were clear with a light background.So the improved Tris-HCl extraction was the most appropriate method for apple leaf SDS-PAGE analysis.