论文

适于SDS-PAGE分析的苹果叶片蛋白质提取方法

  • 曾广娟 ,
  • 李春敏 ,
  • 张新忠 ,
  • 陈东玫 ,
  • 赵永波 ,
  • 董文轩
展开
  • 1. 沈阳农业大学 园艺学院, 辽宁 沈阳 110161;
    2. 河北省农林科学院 昌黎果树研究所, 河北 昌黎 066600
    3. 中国农业大学 园艺植物研究所,北京 100193
曾广娟( 1970- ), 女, 河北吕黎人, 在读博士, 副教授, 主要从事果树种质资源与遗传育种方面研究.

收稿日期: 2009-01-06

  网络出版日期: 2014-10-14

基金资助

河省自然科学基金(C2007000967)

Leaf Protein Extraction Methods Suitable for SDS-PAGE Analysis in Apple

  • ZENG Guang-juan ,
  • LI Chun-min ,
  • ZHANG Xin-zhong ,
  • CHEN Dong-mei ,
  • ZHAO Yong-bo ,
  • DONGWen-xuan
Expand
  • 1. College of Horticulture,Shenyang Agricultural University, Shenyang 110161, China;
    2. Changli Institute of Pomology, Hebei Academy of Agriculture and Forestry Science, Changli 066600, China;
    3. Institute for Horticultural Plants, China Agricultural University, Beijing 100193, China

Received date: 2009-01-06

  Online published: 2014-10-14

摘要

为探索苹果叶片蛋白质SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析的样品制备方法,比较了三氯乙酸(TCA)/丙酮沉淀法、改良的Tris-HC1法、酚-甲醇/醋酸铵沉淀法、二硫苏糖醇(DTT)/丙酮法及Tris-HC1法等5种蛋白质的提取方法.制备的样品经SDS-PAGE分离采用银染显色.结果表明,改良的Tris-HC1法在加大PVPP用量和蛋白质提取缓冲液的基础上,将丙酮沉淀蛋白质的时间由30 min增加到1 h,使蛋白质的得率最高为3.23μg/μL,上样量5μL得到的蛋白质条带数量最多,条带最清晰为1条.利用这一改良方法对苹果实生树不同节位蛋白质的动态变化进行了研究,共发现6条蛋白质差异带,分子量分别为71.9,60.5,52.6,1.1,35.3,18.5 kDa,条带清楚,背景清晰.因此,改良的Tris-HC1法适用于苹果叶片的SDS-PAGE分析.

本文引用格式

曾广娟 , 李春敏 , 张新忠 , 陈东玫 , 赵永波 , 董文轩 . 适于SDS-PAGE分析的苹果叶片蛋白质提取方法[J]. 华北农学报, 2009 , 24(2) : 75 -78 . DOI: 10.7668/hbnxb.2009.02.016

Abstract

In order to develop an efficient protein extraction method suitable for SDS - PAGE analysis,five protocols for protein extraction in apple leaf,trichloroacetic acid/acetone precipitation (TCA),modified Tris - HCl extraction,phenol extraction methanol/ ammonium acetate precipitation,DTT/ acetone and Tris - HCl extraction were compared.The extracts were separated by SDS - PAGE method followed by silver staining. The results showed that the modified Tris-HCl extraction presented the highest resolution of 3. 243μg/μL and the maximum bands of 41 strips with the 5μL sample volumes basing on the increased amount of PVPP and the extracting solution as well as prolonged 30 min of protein precipitation with cold acetone.Dynamic changes of proteins in leaves from different nodes in apple seedlings (Jonathan ×Golden Delicious)were analyzed with the improved Tris - HCl extraction method,six protein bands were detected with the molecular weight 71.9,60.5,52.6,41.1,35.3,18.5 kDa respectively and the bands were clear with a light background.So the improved Tris - HCl extraction was the most appropriate method for apple leaf SDS - PAGE analysis。

Key words: Apple; SDS - PAGE; Extraction

参考文献

[1] Granier F.Extraction of plant proteins for two-dimensional alectrophoresis[J].Electrophoresis,1988,9:712-718.
[2] Gegenheimer P.Preparation of extracts from plants[J].Meth Enzymoi,1990,182:174-193.
[3] Tsugita A,Kamo M.2-D eleetrophoresis of plant proteins[J].Methods Mol Biol,1999,112:95-97.
[4] Damerval C,De Vienne D,Zivy M,et al.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins[J].Electrophoresis,1986,7(1):52-54.
[5] Gorg A,Weiss W,Dunn M J.Current two-dimensional electrophoresis technology for proteomics[J].Proteomics,2004,4:3665-3685.
[6] 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.
[7] Wang W,Scali M,Vignani R,et al.Protein extraction for twodimensional electrophoresis from olive leaf,a plant tissue containing high levels of interfering compounds[J].Electrophoresis,2003,24:2369-2375.
[8] Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[9] Wu F S,Wang M Y.Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues[J].Anal Biochem,1984,139(1):100-103.
[10] Nandaknmar M P,Shen J,Raman B,et al.Solubilization of trichloroacetic acid(TCA)precipitated microbial proteins via NaOH for two-dimensional electrophoresis[J].J Proteome Res,2003,2:89-93.
[11] Molloy M P,Herbert B R,Walsh B J,et al.Extraction of membrane proteins by differential selubilization for separation using two-dimensional gel electrophoresis[J].Electrophoresis,1998,19:837-844.
文章导航

/