搜索

A Non-Radioactive Method for Measuring PP2A Activity in Plants
非放射性法测定植物中的PP2A活性   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Protein phosphatase 2A (PP2A) is a group of important cellular regulators in eukaryotes that dephosphorylate more than 30% of cellular proteins whose activities are turned on or off by phosphorylation. In plants, PP2A was found to regulate critical components involved in plant growth and development, and in response to biotic and abiotic stresses. Therefore, determining the PP2A activities at different developmental stages, in different tissues, or in various mutants is critical in order to understand the functions of PP2A in plants. Traditional PP2A enzyme assay uses radioactive isotope and often take days to finish. This PP2A enzyme assay described here is a method to determine PP2A activity without using radioactive materials in less than 6 h.

Keywords: PP2A activity(PP2A的活性), Plant PP2A(植物PP2A), PP2A holoenzyme(PP2A全酶)

Materials and Reagents

  1. Serine/Threonine Phosphatase Assay Kit (Promega Corporation, catalog number: V2460 )
  2. Protein Assay Dye Reagent (Bio-Rad Laboratories, AbD Serotec®, catalog number: 500-0006 )
  3. Protein Phosphatase (PPase) Inhibitor 2 (I-2) (New England BioLabs, catalog number: P0755S )
  4. Liquid nitrogen (N2)
  5. Tris (pH 7.0) (Thermo Fisher Scientific, catalog number: BP152-5 )
  6. EDTA (Thermo Fisher Scientific, catalog number: S312-12 )
  7. DTT (Sigma-Aldrich, catalog number: D0632 )
  8. Brij 35 (Thermo Fisher Scientific, catalog: BP345-500 )
  9. 1x PP2A assay buffer (see Recipes)

Equipment

  1. Tabletop centrifuge for 96-well plate (Eppendorf, model: 5810R )
  2. Centrifuge at 4 °C or in cold-room (Eppendorf, model: 5415D )
  3. Microplate reader (xMark™ Microplate Absorbance Spectrophotometer) (Bio-Rad Laboratories, catalog number: 1681150 )
  4. Pestle and mortar
  5. 37 °C waterbath

Procedure

  1. Preparation of the desalting column (Note 1)
    1. Pre-wet the spin column provided in the kit with sterile water.
    2. Transfer 10 ml of G25 solution from Serine/Threonine Phosphatase Assay kit to each spin column and let G25 settle by gravity for 5 min.
    3. Wash the column 3 times with 10 ml sterile water, let G25 settle by gravity. Don’t let G25 dry-out.
    4. Wash the column 3 times with 10 ml of 1x PP2A assay buffer, let G25 settle by gravity, seal the column with a cap and proceed to step B1.
    5. Put the column into an empty 50 ml centrifuge tube and spin at 600 x g for 5 min at 4 °C.

  2. Isolate and purification of cellular extracts
    1. Freeze plant materials in liquid N2, if not used immediately, store frozen samples in -80 °C freezer.
    2. Grind frozen plant samples in liquid N2 to fine powder in a pre-cooled mortar and transfer powder to 1.5 ml Eppendorf tubes.
    3. Add 100 µl 1x PP2A assay buffer to each sample (100 mg powder), vortex vigorously to thaw the sample and incubate on ice for 30 min.
    4. Centrifuge at 13,000 rpm for 30 min at 4 °C.
    5. Transfer the supernatant to a fresh tube and incubate the crude extracts on ice briefly.
    6. Proceed to step A5 to prepare the desalting column.
    7. Transfer the supernatant in step B5 to a prepared desalting column in step A5.
    8. Centrifuge the samples at 600 x g for 5 min at 4 °C.
    9. Collect the flow through fluid into fresh tubes and keep them on ice.
    10.  Measure the protein concentration with the Bio-Rad Protein Assay Dye Reagent by mixing 200 µl dye, 800 µl water and 2 µl extracts followed by incubation at room temperature for 5 min before OD595 is recorded, and then normalize the protein concentration to 1 µg/µl with 1x PP2A assay buffer (Protein concentration=6.8*OD595 µg/µl).

  3. Constructing a standard curve for determining the amount of free phosphate
    1. Prepare tubes containing 0, 100, 200, 500, 1,000, 2,000 pmol of phosphates from the 10 mM standard provided in the Serine/Threonine Phosphatase Assay Kit. To ensure the data are comparable, the liquid in all tubes should have a final volume of 50 µl.
    2. Prepare the Molybdate dye/Additive mix (10 µl Additive into 1 ml Molybdate dye) provided by the kit. Use 50 µl Dye/Additive for each tube and mix by vortexing.
    3. Centrifuge at 13,000 rpm at room temperature for 30 sec.
    4. Carefully transfer the supernatants to the 96-well plate provided by the kit while avoiding air-bubbles.
    5. Centrifuge the 96 well plate for 2 min followed by incubation at room temperature for 5 min.
    6. Read the optical density with a plate reader using a 600 nm filter.
    7. Prepare the standard curve based on the OD600 readings and the pmol phosphates used in the assay (Note 5).

  4. The PP2A activity assay
    1. Prepare the Molybdate dye/Additive just before the assay starts. Use 50 µl Dye/Additive for each PP2A assay. Calculate the amount needed for the experiment and only prepare sufficient fresh solution for each assay (Note 6).
    2. Mix the PP2A assay components on ice.
      Components
      Amount
      Purified extracts
      5 µl
      Peptide substrates
      5 µl
      10x PP2A assay buffer
      5 µl
      PPase inhibitor 2
      1 µl
      Phosphate free water
      34 µl
    3. Incubate the reaction mixture at 37 °C for 5 min.
    4. Terminate the reaction by adding 50 µl Dye/Additive.
    5. Mix well and centrifuge at 13,000 rpm at room temperature for 1 min.
    6. Transfer the mixture to a 96-well plate and avoid air-bubbles (Note 7).
    7. Allow the plate to incubate at room temperature for 15 min for color development (Note 8).
    8. Read the optical density with a plate reader using a 600 nm filter.

  5. Calculation of PP2A activity
    1. Based on the standard curve, determine the amount of phosphate released from the reaction.
    2. Use the following equation to determine the PP2A activity.
      PP2A activity = amount of phosphate released (pmol)/5 (min)/5 (µg).
    3.  The PP2A activity unit is defined as pmol phosphate released from substrates per min per µg total protein. The PP2A activity should be around 55-60 units for cellular extracts from wild-type Columbia Arabidopsis plants.
    4. Technical replicates and biological replicates should be performed to obtain the variance between experiments.

Representative data



Figure 1. The PP2A activity of WT and two plants (amiR-6 & amiR-4) containing artificial microRNAs designed to down regulate AtPTPA. A Typical assay for WT Arabidopsis has PP2A activity of 55~60 units. amiR plants have reduced PP2A activity due to the degradation of mRNA of AtPTPA, which is essential for PP2A holo-enzyme assembly (see Chen et al., 2014 for detail).

Notes

  1. It is important that the protein samples are desalted in order to remove the free phosphate that can interfere with the measurements of the phosphate released by PP2A.
  2. Samples need to be completely thawed and mixed well before centrifugation at 4 °C.
  3. Avoid transferring debris, as it will clog the desalting column.
  4. Use x g force in centrifugation; however, if using microfuge, rpm is used in desalting the crude extracts, higher speed gives varied desalting efficiency and sample yield.
  5. Construct a new standard curve every month after the kit is opened. Please refer the short manual included in this kit for more information about the standard curve. Link to the manual.
  6. All the assay component should be stored at 4 °C. Never freeze the peptide substrates and the kit should be stable for 3 months after it is opened.
  7. Spin the 96-well plate again to remove the air bubble if bubble occurs.
  8. If higher amount of protein (greater than 5 µg) is used for the assay, allow more time for color development. Be sure that the amount of PP2A in the extract does not deplete the phosphate group from the peptide substrates. Perform a titration experiment to determine a linear range of reactions, so the actual assay can be performed within linear range of reactions.

Recipes

  1. 1x PP2A assay buffer
    50 mM Tris (pH 7.0)
    0.1 mM EDTA
    5 mM DTT and 0.01% Brij 35

Acknowledgments

This work was supported by the Department of Biological Sciences and the Graduate School of Texas Tech University, by the National Natural Science Foundation of China (grant no. 31170793 to Guoxin Shen), and by the National Natural Science Foundation of Zhejiang Province (grant no. Z12c130011 to Guoxin Shen). The protocol is adapted from Chen et al. (2014).

References

  1. Chen, J., Hu, R., Zhu, Y., Shen, G. and Zhang, H. (2014). Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR is essential for PROTEIN PHOSPHATASE 2A holoenzyme assembly and plays important roles in hormone signaling, salt stress response, and plant development. Plant Physiol 166(3): 1519-1534.

简介

蛋白磷酸酶2A(PP2A)是一组重要的细胞调节剂在真核生物中去磷酸化超过30%的细胞蛋白质,其活性通过磷酸化打开或关闭。 在植物中,PP2A被发现调节涉及植物生长和发育以及响应生物和非生物胁迫的关键组分。 因此,为了理解PP2A在植物中的功能,确定在不同发育阶段,在不同组织或在各种突变体中的PP2A活性是至关重要的。 传统的PP2A酶测定使用放射性同位素,并且通常需要几天才能完成。 这里描述的PP2A酶测定法是在不使用放射性材料的情况下在少于6小时内测定PP2A活性的方法。

关键字:PP2A的活性, 植物PP2A, PP2A全酶

材料和试剂

  1. 丝氨酸/苏氨酸磷酸酶测定试剂盒(Promega Corporation,目录号:V2460)
  2. 蛋白质测定染料试剂(Bio-Rad Laboratories,AbD Serotec ,目录号:500-0006)
  3. 蛋白磷酸酶(PPase)抑制剂2(I-2)(New England BioLabs,目录号:P0755S)
  4. 液氮(N 2)
  5. Tris(pH7.0)(Thermo Fisher Scientific,目录号:BP152-5)
  6. EDTA(Thermo Fisher Scientific,目录号:S312-12)
  7. DTT(Sigma-Aldrich,目录号:D0632)
  8. Brij 35(Thermo Fisher Scientific,目录:BP345-500)
  9. 1x PP2A测定缓冲液(见配方)

设备

  1. 用于96孔板(Eppendorf,型号:5810R)的台式离心机
  2. 在4℃或冷室中离心(Eppendorf,型号:5415D)
  3. 微孔板读数器(xMark TM微孔板吸光度分光光度计)(Bio-Rad Laboratories,目录号:1681150)
  4. 杵和臼
  5. 37°C水浴

程序

  1. 脱盐柱的准备(注1)
    1. 用无菌水预湿试剂盒中提供的旋转柱
    2. 从丝氨酸/苏氨酸磷酸酶测定转移10ml G25溶液 试剂盒到每个旋转柱,并让G25通过重力沉降5分钟
    3. 用10ml无菌水洗涤柱3次,使G25通过重力沉降。 不要让G25变干。
    4. 用10ml 1×PP2A测定缓冲液洗涤柱3次,使G25 通过重力沉降,用盖子密封柱并继续步骤B1
    5. 将柱放入空的50ml离心管中,并在4℃下以600×g旋转5分钟。

  2. 细胞提取物的分离和纯化
    1. 冻结液体N 2中的植物材料,如果不立即使用,将冷冻样品储存在-80℃冰箱中。
    2. 在预冷却的砂浆中将冷冻植物样品在液体N 2中研磨成细粉末,并将转移粉末转移到1.5ml Eppendorf管中。
    3. 向每个样品(100 mg粉末)中加入100μl1×PP2A测定缓冲液,涡旋 大力解冻样品并在冰上孵育30分钟
    4. 在4℃下以13,000rpm离心30分钟
    5. 转移上清液到一个新鲜的试管,并短暂孵育粗提取物在冰上。
    6. 继续执行步骤A5以准备脱盐柱。
    7. 将步骤B5中的上清液转移到步骤A5中制备的脱盐柱
    8. 在4℃下以600×g离心样品5分钟
    9. 通过流体将流体收集到新管中并保持在冰上
    10.  使用Bio-Rad Protein Assay Dye测量蛋白质浓度 试剂通过混合200μl染料,800μl水和2μl提取物,然后   在室温下孵育5分钟,然后记录OD 595 然后用1x PP2A测定法将蛋白质浓度标准化为1μg/μl 缓冲液(蛋白质浓度= 6.8 * OD595μg/μl)。

  3. 构建标准曲线以确定游离磷酸的量
    1. 准备含有0,100,200,500,1,000,2,000pmol的试管 来自丝氨酸/苏氨酸中提供的10mM标准的磷酸盐 磷酸酶测定试剂盒。 为了确保数据是可比的,液体中 所有试管的最终体积应为50μl
    2. 准备 钼酸盐染料/添加剂混合物(10μl添加剂到1ml钼酸盐染料中) 由套件提供。 使用50微升染料/添加剂为每个管和混合 涡旋
    3. 在室温下以13,000rpm离心30秒
    4. 小心地将上清液转移到试剂盒提供的96孔板,同时避免气泡。
    5. 离心96孔板2分钟,然后在室温下孵育5分钟
    6. 使用600 nm过滤器用读板器读取光密度
    7. 基于OD 600读数和测定中使用的pmol磷酸盐制备标准曲线(注5)。

  4. PP2A活性测定
    1. 在测定开始前准备钼酸盐染料/添加剂。 使用 每个PP2A测定50μl染料/添加剂。 计算所需的金额 实验并且仅为每个测定制备足够的新鲜溶液   (注6)。
    2. 在冰上混合PP2A测定组分。
      组件
      金额
      纯化提取物
      5微升
      肽底物
      5微升
      10x PP2A测定缓冲液
      5微升
      PPase抑制剂2
      1微升
      无磷水
      34微升
    3. 将反应混合物在37℃下孵育5分钟
    4. 通过加入50μl染料/添加剂终止反应
    5. 充分混合并在室温下以13,000rpm离心1分钟
    6. 将混合物转移到96孔板,避免气泡(注7)。
    7. 让板在室温下孵育15分钟以进行显色(注8)
    8. 使用600 nm过滤器用读板器读取光密度

  5. 计算PP2A活性
    1. 基于标准曲线,确定从反应中释放的磷酸盐的量
    2. 使用以下公式确定PP2A活性 PP2A活性=释放的磷酸盐的量(pmol)/5(min)/5(μg)
    3.   PP2A活性单位定义为从释放的磷酸pmol 底物每分钟每μg总蛋白。 PP2A活性应为 约55-60单位的来自野生型哥伦比亚的细胞提取物 拟南芥植物
    4. 应进行技术复制和生物复制以获得实验之间的差异。

代表数据



图1.含有设计用于下调AtPTPA的人工微RNA的WT和两种植物(amiR-6和amiR-4)的PP2A活性。对于WT拟南芥的典型测定具有55〜60的PP2A活性单位。由于AtPTPA的mRNA的降解,amiR植物具有降低的PP2A活性,这对于PP2A全酶聚集是必需的(详见Chen等人,2014年)。

笔记

  1. 重要的是,蛋白质样品脱盐以除去可干扰由PP2A释放的磷酸盐的测量的游离磷酸盐。
  2. 在4℃下离心之前,样品需要完全融化并充分混合
  3. 避免转移碎屑,因为它会堵塞脱盐柱。
  4. 在离心时使用 x g 力;然而,如果使用微量离心,rpm用于脱盐粗提取物,更高的速度提供不同的脱盐效率和样品产率。
  5. 打开套件后,每月建立一个新的标准曲线。有关标准曲线的更多信息,请参阅本套件中的简要手册。 链接到手册。
  6. 所有测定组分应储存在4℃。切勿冷冻肽底物,试剂盒在打开后应稳定3个月。
  7. 如果发生气泡,再次旋转96孔板以除去气泡
  8. 如果较高量的蛋白质(大于5μg)用于测定,则允许更多的时间用于显色。确保提取物中PP2A的量不会耗尽肽底物上的磷酸基团。执行滴定实验以确定反应的线性范围,因此实际测定可以在反应的线性范围内进行。

食谱

  1. 1x PP2A测定缓冲液
    50mM Tris(pH7.0)
    0.1mM EDTA
    5mM DTT和0.01%Brij 35

致谢

这项工作由生物科学系和德克萨斯理工大学研究生院,中国国家自然科学基金(授予国家沉阳)和浙江省国家自然科学基金资助 .Z12c130011 to Guoxin Shen)。 该协议改编自Chen等人(2014)。

参考文献

  1. Chen,J.,Hu,R.,Zhu,Y.,Shen,G.and Zhang,H。(2014)。 拟南芥磷酸肌醇磷酸酶激活剂是蛋白磷酸酶2A全酶组装和播放的必需品 在激素信号转导,盐胁迫反应和植物发育中的重要作用。植物生理学166(3):1519-1534。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, J., Shen, G. and Zhang, H. (2015). A Non-Radioactive Method for Measuring PP2A Activity in Plants. Bio-protocol 5(17): e1577. DOI: 10.21769/BioProtoc.1577.
  2. Chen, J., Hu, R., Zhu, Y., Shen, G. and Zhang, H. (2014). Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR is essential for PROTEIN PHOSPHATASE 2A holoenzyme assembly and plays important roles in hormone signaling, salt stress response, and plant development. Plant Physiol 166(3): 1519-1534.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。