秋茄根系蛋白质组的双向电泳技术优化

Optimization of Two-dimensional Polyaneylamide Gel Electrophoresis for Proteome of Kandelia candel Roots

建立适用于秋茄根系蛋白质组分析的双向电泳技术,并对秋茄根系蛋白质的制备、溶解、上样量、IEF及SDS-PAGE电泳等关键步骤进行优化。结果表明,应用酚抽法来提取秋茄根系蛋白,更适用于双向电泳分析;秋茄根系蛋白主要分布在pH4～7范围;裂解液中含有硫脲(2mmol.L-1)能较充分地溶解蛋白,DTT浓度为40mmol.L-1、上样量1.5mg时得到的双向电泳图谱分辨率较好,且蛋白斑点分布均匀、清晰,拖尾现象明显减少。采用与质谱兼容的考马斯亮兰进行染色,得到1 089个蛋白点。

A two-dimensional polyarelamide gel electrophoresis（2D-PAGE） system was established for proteomic analysis of Kandelia candel roots.Protein extraction,dissolution,loading quantity,IEF and SDS-PAGE parameters in the two-dimensional electrophoresis（2-DE） were optimized.The results showed that phenol method was suitable for the root protein extraction,the protein isoelectric points mainly distributed between pH 4 and 7.The lysis buffer with 2 mmol·L-1 thiourea improved dissolution of protein effectively and the resolution of spectrum for the dissolved protein was increased with 40 mmol·L-1 DTT and 1.5 mg loading amount.The 1089 protein spots were stained with Colloidal coomassie blue and detected by mass spectrometry.