ZHANG Yong,SHI Ting,WU Xinxin,et al.Establishment and optimization of Western-blot system for CCoAOMT protein in Prunus mume[J].Journal of Nanjing Agricultural University,2017,40(6):977-982.[doi:10.7685/jnau.201702012]





Establishment and optimization of Western-blot system for CCoAOMT protein in Prunus mume
张勇 侍婷 吴欣欣 高志红
南京农业大学园艺学院, 江苏 南京 210095
ZHANG Yong SHI Ting WU Xinxin GAO Zhihong
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
Prunus mumeflower budprotein extractioncaffeoyl-coA-O-methyl transferase(CCoAOMT)Western-blot
[目的] 本文旨在建立一套适合于果梅(Prunus mume) Western-blot的蛋白质提取和技术体系。[方法] 以果梅品种‘小绿萼’花芽为试验材料,以咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)为目标蛋白,分别采用Tris-HCl法和SDS上样缓冲液直接提取法,比较2种提取方法的优劣,并通过设置不同的蛋白上样量(20、30、40、50及60 μg)、转膜时间(40、60及90 min)、一抗稀释比例(1:500、1:1 000、1:2 000及1:5 000)以及一抗孵育时间(室温1 h、室温2 h及4℃过夜)等组合,研究适合果梅花芽CCoAOMT蛋白Western-blot的最佳体系。[结果] 成功建立了快速、灵敏、特异的Western-blot体系,确定了果梅花芽CCoAOMT蛋白的Western-blot试验条件。SDS上样缓冲液直接提取法提取蛋白快速、高效,最佳条件为以蛋白上样量30 μg、转膜1 h、一抗稀释比例1:1 000以及一抗孵育2 h。[结论] 本试验建立了简单、快速和重复性好的SDS上样缓冲液直接提取法以及优化的上样量、转膜时间和抗体浓度等Western-blot体系,为梅等木本植物开展Western-blot研究提供依据。
[Objectives] The paper aims to establish the method which is appropriate for protein extraction and Western-blot analysis of Prunus mume.[Methods] We used flower buds of Prunus mume ‘Xiaolüe’ as the plant material and PmCCoAOMT as the target protein. Tris-HCl method and SDS loading buffer method were used to extract flower bud protein, respectively. Furthermore, we analyzed advantages and disadvantages of two methods by comparing Tris-HCl method with SDS loading buffer method. In order to study the optimum Western-blot system for CCoAOMT protein of Japanese apricot, different weights of protein samples (20, 30, 40, 50 and 60 μg), transmembrane time (40, 60 and 90 min), primary antibody concentration (1:500, 1:1 000, 1:2 000 and 1:5 000)and incubation time of primary antibody (at room temperature for 1 h, at room temperature 2 h and at 4℃ for overnight)were tested.[Results] The rapid, sensitive and specific Western-blot system has been established successfully, and the experimental Western-blot condition of CCoAOMT protein in the flower bud of Japanese apricot was determined. SDS loading buffer extraction method was more rapid and efficient. The optimal conditions were weight of protein samples (30 μg), transmembrane time (1 h), primary antibody concentration (1:1 000)and incubation time of primary antibody (2 h).[Conclusions] The SDS loading buffer direct extraction method is simple, rapid and reproducible, which provides the basis for the extraction of woody plant protein and the research of Western-blot.


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更新日期/Last Update: 1900-01-01