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[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

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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.

简介

植物根秘密很多过氧化物酶抵抗环境影响。 这个协议描述了一种从拟南芥植物提取根体外形成壁液体和使用愈创木酚作为底物确定过氧化物酶活性的方法。

关键字:过氧化物酶, 根, 愈创木酚, 质外体液流, 酶活

材料和试剂

  1. 10mM Na + + -PO 4(pH 6.0)
  2. Guaiacol
  3. 琼脂
  4. MES-K
  5. 1/4x改良的Hoagland培养基(参见用于配方的一般生长方案)
  6. 固体介质(见配方)

设备

  1. UV-vis光谱仪
  2. 拟南芥生长设施
  3. 15 ml Falcon管

程序

  1. 植物生长
    表面灭菌的拟南芥种子种植在 x修饰的Hoagland培养基(1%琼脂,5mM MES-K,pH 6.0)在1ml移液管吸头盒插入物(具有孔的部分)上固化。 在光照下萌发2-3天后,将具有幼苗的箱子漂浮在 改良的Hoagland液体培养基与5mM MES-K,pH 6.0,通过鱼缸水泵充气。
  2. 外形壁液(AWF)的提取
    1. 将Col幼苗(2.5周龄)的根在去离子水中漂洗,并切成10mM Na + + -PO 4(pH 6.0)。
    2. 在冰上真空5分钟,然后缓慢释放用于渗透。
    3. 然后将根切到滤纸上干燥,在天平上测量并记录为鲜重。
    4. 然后将根部放置到5ml注射器的插入筒中。 将注射器筒置于15ml Falcon管中并在3,000℃下在4℃下离心15分钟。
    5. AWF从管底部救出。 通过移液测量总体积。 AWF保持在冰上。
  3. 总蛋白质定量
    蛋白质浓度测量(Bradford微量测定)根据Bio-Rad试剂说明书进行 要设置反应,请混合以下内容:
    化学体积
    愈创木酚880μl124.14; D = 1.112g/ml,20mM,在10mM Na-PO 4 pH 6.0中 AWF x微博
    加入20-xμl10mM Na-PO 4(pH 6.0) 通过添加:
    开始反应 H 2 2 O 2100μl34.01; 31.4%0.3%(v/v)
    总体积调整为1ml。
    反应在室温下在470nm分光光度法进行2分钟。消光系数= 26.6/Mm/cm 2 / > > /b>
    图1。
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引用:Li, X. (2011). Extraction of Root Apoplastic Wall Fluid for Apoplastic Peroxidase Activity Assay. Bio-protocol Bio101: e127. DOI: 10.21769/BioProtoc.127;
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