欢迎您, 登录 | 注册

首页 | English

X
加载中

Plant roots secret a lot of peroxidases to counteract environmental influences. This protocol describes a way to extract root apoplastic wall fluid from Arabidopsis plants and to determine the peroxidase activity using guaiacol as substrate.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Extraction of Root Apoplastic Wall Fluid for Apoplastic Peroxidase Activity Assay
[Bio101] 根壁外非原质体液的提取及非原质体过氧化物酶活性分析

植物科学 > 植物细胞生物学 > 细胞结构
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
9/5/2011, 7276 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.127

[Abstract] Plant roots secret a lot of peroxidases to counteract environmental influences. This protocol describes a way to extract root apoplastic wall fluid from Arabidopsis plants and to determine the peroxidase activity using guaiacol as substrate.

Keywords: Peroxidase(过氧化物酶), Root(根), Guaiacol(愈创木酚), Apoplastic fluid(质外体液流), Enzyme activity(酶活)

Materials and Reagents

  1. 10 mM Na+-PO4 (pH 6.0)
  2. Guaiacol
  3. Agar
  4. MES-K
  5. 1/4x modified Hoagland medium (see general growth protocol for recipes)
  6. Solid medium (see Recipes)

Equipment

  1. UV-vis Spectrometer
  2. Arabidopsis growth facility
  3. 15 ml Falcon tube

Procedure

  1. Plant growth
    Surface-sterilized Arabidopsis seeds are planted on ? x modified Hoagland medium (1 % agar, 5 mM MES-K, pH 6.0) solidified on 1 ml pipette tip box insert (the part with holes). After germination for 2-3 days under light, the boxes with young seedlings are floated in ? modified Hoagland liquid medium with 5 mM MES-K at pH 6.0, aerated by fish tank water pumps.
  2. Extraction of apoplastic wall fluid (AWF)
    1. Roots of Col seedlings (2.5 weeks old) are rinsed in de-ionized water and cut into 10 mM Na+-PO4 (pH 6.0).
    2. Vacuum for 5 min on ice, followed by slow release for infiltration.
    3. The roots are then dabbed onto filter paper to dry, measured on a balance and recorded as fresh weight.
    4. The roots are then put to the plunge barrel of a 5 ml syringe. The syringe barrel is placed in a 15 ml Falcon tube and centrifuged at 3,000 x g for 15 min at 4 °C. 
    5. The AWF is rescued from the tube bottom. Total volume is measured by pipetting. AWF is kept on ice.
  3. Total protein quantification
    Protein concentration measurement (Bradford micro assay) is done according to Bio-Rad reagent instruction.
    To set up a reaction, mix the following:
    Chemical Volume FW Stock conc.
    Guaiacol 880 μl 124.14; D=1.112 g/ml, 20 mM in 10 mM Na-PO4 pH 6.0
    AWF x μl
    Add 20-x μl 10 mM Na-PO4 (pH 6.0).
    Start the reaction by adding:
    H2O2 100 μl 34.01; 31.4% 0.3% (v/v)
    Total volume is adjusted to 1 ml.
    Reactions are followed spectrophotometrically at 470 nm for 2 min at room temperature. Extinction coefficient = 26.6/Mm/cm

    Figure 1.


How to cite this protocol: Li, X. (2011). Extraction of Root Apoplastic Wall Fluid for Apoplastic Peroxidase Activity Assay. Bio-protocol Bio101: e127. DOI: 10.21769/BioProtoc.127; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册