不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

Effects of Different Protein Extraction Methods and Loading Amount on 2-DE Profile for Basal Part of Mudan in vitro Stem

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPGpH3～10,24cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。

In this paper,we compared the effects of different protein extraction methods and loading amount on 2-DE profile in order to establish a suitable and efficient 2-DE technique for proteome of basal part of stem of mudan in vitro and facilitate rooting mechanism research by using proteome approach. The results showed that samples prepared by ammonium acetate/methanol-phenol extraction only got 45 protien spots on the 2-DE profile,and the spots were indistinct,had obvious streak appearance and low resolution. Samples obtained by method of ethanol/ether-acetone precipitation got 101 protien spots that were not clear either and had an obvious interference of crosswise and upright streaks. While samples acquired by TCA/acetone precipitation had more distinguishable and regularly-shaped protein spots （about 434）,which was of good reproducibility and was suitable for subsequent analysis. And also TCA/acetone precipitation approach was more convenient for manipulation. Furthermore,we employed different loading amount proteins extracted by the method of TCA/acetone for 2-DE,that is,800 μg,1 000 μg and 1 200 μg. Proteins of 1 200 μg loading amount separated well on the 12% SDS-PAGE gel with IPG gel of pH 3～10,24 cm,562 protein spots were distinguished on the 2-DE profile. As a result,TCA/acetone precip-itation was much more suitable for protein extraction of basal part of stem of mudan in vitro and 1 200 μg was the optimum loading amount.