一种快速的水稻不同组织总蛋白提取方法

苏军; 张武君; 陈在杰

doi:10.19303/j.issn.1008-0384.2013.09.010

一种快速的水稻不同组织总蛋白提取方法

doi: 10.19303/j.issn.1008-0384.2013.09.010

福建省农业遗传工程重点实验室/福建省农业科学院生物技术研究所,福建福州,350003

基金项目:

福建省财政专项——福建省农业科学院科技创新团队建设项目 (CXTD2011-08)

福建省自然科学基金项目 (2011J01105)

国家自然科学基金项目 (30771298)

详细信息

作者简介:
苏军 (1961-), 女, 博士, 研究员, 主要从事植物基因工程技术研究

中图分类号: S511
计量
- 文章访问数: 259
- HTML全文浏览量: 50
- PDF下载量: 16
- 被引次数: 0
出版历程
- 收稿日期: 2013-05-31
- 刊出日期: 2013-09-18

A Simple and Rapid Applicable Protocol for Extracting High-quality Total Proteins from Rice

Fujian Provincial Key Laboratory of Genetic Engineering for Agriculture/Biotechnology Institute of Fujian Academy of Agricultural Sciences

摘要

摘要: 介绍一种简单快捷的水稻不同组织总蛋白提取方法。该方法采用预冷甲醇和丙酮依次洗涤去除杂质和次生代谢干扰物, 同时在甲醇洗涤步骤中加入蛋白酶抑制剂混合液, 能够最大限度地包含所有蛋白质并降低蛋白酶的降解作用, 提高蛋白质的质量和产量。采用该方法从水稻的根、茎、叶及发育中的种子中提取粗蛋白, 产量为20~99mg·g-1FW, 提取蛋白整个过程耗时1.6h。以SDS-PAGE胶及Western Blot检测所提总蛋白的质量, 分别用核蛋白、胞浆蛋白、膜蛋白以及淀粉结合型蛋白等不同类型蛋白抗体进行Western Blot检验。结果显示采用该方法所分离的总蛋白质量可以满足水稻不同类型蛋白对Western Blot的质量要求。
- 水稻 /
- 总蛋白提取 /
- WesternBlot
Abstract: A simple and rapid applicable protocol for extracting high-quality proteins from rice tissues is described.In the protocol, precooling methanol and acetone is used to precipitate proteins and remove contaminants and secondary metabolites.Adding protease inhibitors in methanol for washing is include all proteins to maximize and reduce degradation of proteases.By using this protocol the total protein yield of different tissues were range from 20 to 99mg·g-1FW and the entire process taken 1.6h.To evaluate the quality of the extracted protein, SDS/PAGE gel and Western Blot were employed.4 antibodys represented different protein type include the nucleoprotein antibody, plasmosin antibody, membrane protein antibody and granule-bound starch protein antibody as first antibody for Western Blot.The results show the extraction of high-quality protein samples suitable for Western analysis, and the overall quality of the protein profiles was good.The simplified protocol described here presents considerable savings of time and reagents.
- rice /
- total protein extract /
- western blot

HTML全文

参考文献(12)

[1]	JOCELYN K C R, SAJID B, JAMES J G, et al.Tackling the plant proteome:practical approaches, hurdles and experimental tools[J].The Plant Journal, 2004, 39 (5) :715-733.
[2]	BRADFORD M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Biochem, 1976, 72:248-254.
[3]	谷瑞升, 刘群录, 陈雪梅, 等.一种省时高效的木本植物蛋白双向电泳分析方法[J].北京林业大学学报, 1999, 21 (5) :7-10.
[4]	应雯, 陆徐忠, 李莉, 等.适于蛋白双向电泳的水稻叶片样品提取方法初探[J].生物学杂志, 2010, 27 (3) :84-87.
[5]	DAMERVAL C, VIENNE D D, ZIVY M, et al.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seed-ling proteins[J].Electrophoresis, 1986, 7:52-54.
[6]	HURKMAN W J, TANAKA C K.Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis[J].Plant Physiol, 1986, 81:802-806.
[7]	康俊梅.低温胁迫下野牛草生理生化响应及蛋白质组学研究[D].北京:中国农业科学院, 2008:44-50.
[8]	YAO Y, YANG Y W, LIU J Y.An efficient protein preparation for proteomic analysis of developing cotton fibers by2-DE[J].Electrophoresis, 2006, 27:4559-4569.
[9]	HERMAN E M, HELM R M, JUNG R.Genetic modification removes an immunodominant allergen from soybean[J].Plant Physiology, 2003, 132:36-43.
[10]	陈晶瑜, 郭宝峰, 何付丽, 等.适合双电泳的植物全蛋白提取方法比较[J].中国农学通报, 2010, 26 (23) :97-100.
[11]	王玉琪, 彭新洲.适用于水稻叶片蛋白质组分析的双向电泳技术[J].植物生理与分子生物学报, 2006, 32 (2) :252-256.
[12]	SEDDON A M, CURNOW P, BOOTH P J.Membrane proteins, lipids and detergents:not just a soap opera[J].Biochim Biophys Acta, 2004, Nov 3:1666 (1-2) :105-107.