The improvement of the protein profiling of Ailanthus altissima pollen extract using highlength immobilized pH gradient as the first dimension for 2-DE

Two-D gel electrophoresis (2DE) is known as a powerful technique for the separation of individual proteins from complex samples based on their isoelectric points and molecular weights. This technique expands the number of proteins that could be identified. Recently, coupling 2-DE with immobilized pH gradients has provided higher resolution of this method. The aim of this study was the improvement of the protein profiling of Ailanthus altissima pollen extract using 18 cm IPG strips (pH 3–10 nonlinear). For this purpose, the pollen proteins were extracted in phosphate-buffered saline (PBS). Dialysis is used to remove salts from protein solutions. The protein content of A. altissima pollen extract was studied using the Bradford protein assay followed by a 2-DE. Approximately 400 μg A. altissima protein extracts wasadded to the sample rehydration buffer. Results showed that the total protein concentration of AP extract was 4.3 μg per μl. Two-D electrophoresis of AAP extracts revealed 400 protein spots distributed in a wide range of pI and molecular masses. However, our previous study using 7 cm IPG strips (pH 3–10 nonlinear) allowed the detection of only 125 protein spots. Therefore, IPG strip length and gel dimensions significantly influence the resolution and sample throughput on two-dimensional gels and proven experience the maximal resolution is obtained using 18 and 24 cm IPG strips.

کلیدواژه ها:

2D gel electrophoresis ، Strip ، Protein ، Dialysis ، Pollen ، Protein profile