家蚕蛋白质双向电泳的样品制备方法

进行蛋白质组研究的关键是要尽可能完整地获得一个基因组在一个生物体或一个组织器官的特定时期表达的蛋白质的种类和数量 ,双向电泳技术是分离组织和器官蛋白质的核心技术 ,而蛋白质样品的制备是双向电泳的基础。以家蚕胚胎、中肠、皮肤、丝腺等组织器官为材料 ,采用磷酸缓冲液或Tris HCl缓冲液抽提蛋白质样品 ,用 6种不同的蛋白质溶解缓冲液溶解蛋白质 ,经蛋白质双向电泳和蛋白质图像软件分析 ,结果表明用磷酸缓冲液抽提、蛋白质溶解缓冲液E溶解是制备家蚕蛋白质双向电泳样品的较好方法。蛋白质磷酸抽提缓冲液 (PBS)的组成为 :32 5mmol/LK2 HPO4,2 6mmol/LKH2 PO4,4 0 0mmol/LNaCl,pH 7 6。蛋白质样品溶解缓冲液E的组成为 :8mol/L尿素 ,2mol/L硫尿 ,4 %CHAPS ,2 0mmol/LTrisbase,30mmol/LDTE ,2 %Pharmalyte(pH 3～ 10 )。

Preparation of Silkworm Protein Using Two Dimensional Polyacryamide Gel Electrophoresis

The key to research on the protein is to gain the complete variety and amounts of the protein as much as possible, the protein expressed by the genome in a life or an organ in the specific stage. The 2D-PAGE (two dimensional polyacryamide gel electrophoresis) is the key skill to isolate the protein from the tissues and organs, and the extraction of the protein sample is the base of 2D-PAGE. The proteins ware extracted from the silkworm (Bombyx mori L.) embryo, midgut, skin, silk gland and other organs by phosphate buffer or Tris-HCl buffer. The pellet was suspended in six different kinds of lysis buffer respectively. The results indicated that it is a better way to prepare silkworm protein by 2D-PAGE with phosphate buffer as protein extraction buffer and the protein lysis buffer E as lysis buffer. The composition of the phosphate buffer is as follows: 32.5 mmol/L K 2HPO 4,2.6 mmol/L KH 2PO 4,400 mmol/L NaCl, pH 7.6. The composition of the protein lysis buffer E is as follows: 8 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 20 mmol/L trisbase, 30 mmol/L DTE, 2% pharmalyte pH 3～10.