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MAPK Phosphorylation Assay with Leaf Disks of Arabidopsis
拟南芥叶片中MAPK的磷酸化检测

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Abstract

Activation of mitogen activated protein kinases (MAPKs) is involved in many abiotic and biotic stress responses including plant defense. MAPK acitvation is based on the dual phosphorylation of threonine (T) and tyrosin (Y) residues (T-x-Y motif) in the activation loop of the MAPK protein. By determination of the phosphorylation status of a specific MAPK one can detect if the MAPK has been activated or not.
This protocol describes how to analyze the phosphorylation status of Arabidopsis MAPKs MPK3 and MPK6 by using leaf disks, western blotting and a specific antibody (Figure 1). It can also be used for the analysis of MAPKs in other plant systems although some alterations regarding protein extraction might be necessary.

Keywords: Arabidopsis(拟南芥), Kinase(激酶), PTI(PTI), Innate Immunity(先天免疫), Microbe(微生物)



Figure 1. Detection of the phosphorylation of Arabidopsis thaliana MAPKs MPK6 and MPK3 upon treatment of seedlings for 15 min with the active epitope (flg22, 1 μM) of the bacterial elicitor flagellin (+). No phosphorylated MAPKs were detected in the control treated sample (-).

Materials and Reagents

  1. Arabidopsis thaliana adult plants
  2. Liquid nitrogen
  3. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb #4370 (Cell Signaling Technology) (http://www.cellsignal.com/products/4370.html)
  4. Anti-Rabbit secondary antibody
  5. Tris-HCl
  6. Glycerol
  7. SDS
  8. Dithiothreitol (DTT)
  9. Bromphenol blue
  10. Reagents for SDS-PAGE and Western Blotting
  11. 6x Protein extraction/loading buffer (see Recipes)

Equipment

  1. 1.5 ml Microcentrifuge tubes
  2. Small petri dishes (~ 5 cm diameter) or 6-well plates
  3. Cork borer (~ 10 mm diameter)
  4. Glass beads (~ 1-2 mm diameter)
  5. Silamat S6 (http://www.ivoclarvivadent.com/en/products/equipment/mixer/silamat-s6)
  6. Alternatively for 4. and 5. Mortar (~ 5 cm diameter) and pestle
  7. Vortexer
  8. Heating block
  9. Centrifuge
  10. Equipment for SDS-PAGE, Western Blotting and detection

Procedure

  1. Leaf disks are cut from adult Arabidopsis (Arabidopsis thaliana) leaves using the cork borer. Try to take leaves of similar age and fitness but from different plants.
  2. Weigh one exemplary leaf disk and note the weight (usually ~ 20 mg).
  3. Float disks on distilled water in small petri dishes or 6-well plates (at least 3 per dish or well in approximately 5 ml of water).
  4. Wait overnight (usually 16 h, dishes closed) and keep the dishes in an undisturbed and controlled place (e.g. growth chamber of origin). This will lead to dephosphorylation of MAPKs which have been phosphorylated upon harvesting.
  5. Next morning perform the treatment, for example add an elicitor peptide of which you would like to know if it triggers MAPK phosphorylation. We add it to a final concentration of 1 μM, mix carefully to not mechanically stress the leaf tissue and in most cases wait for 15 min. As a proper control use a mock treatment (e.g. solvent only) and also incubate for 15 min. This is critical, since MAPK phosphorylation is triggered very easily by many different stresses. Thus you need to show that your control treatment does not lead to phosphorylation of MAPKs. Other treatments could be addition of chemicals, wounding stress, UV-treatment etc.
    Note: Phosphorylation and activation of MAPKs happens very quickly and is transient. It starts roughly 2-5 min after treatment and lasts at least for 1 h or longer.
  6. Harvest all disks from one well after the desired incubation time quickly with a forceps and dry them briefly on a paper towel (residual water will dilute your sample and interfere with the grinding procedure). Place them into a microcentrifuge tube (preferably safe lock) that contains 5 glass beads and shock-freeze them in liquid nitrogen. Repeat this procedure for each well.
    Note: Here you have to be quick! Harvesting the disks (mechanical stress) will induce MAPK phosphorylation, thus you should not take longer than 1 min for each sample to avoid false positive phosphorylation results!
  7. Grind tissue to fine powder (the tissue should not thaw before the next step!). You can do this by using a mortar and pestle (cooled in liquid nitrogen) but we do not recommend this. Especially if you have many samples grind the tissue by using a Silamat S6 (or similar machine) that grinds the tissue in the tube due to rapid movement and the previously added glass beads.
  8. Add extraction/loading buffer. For example for 60 mg of tissue, use 60 μl and use the same ratio depending on sample weight.
  9. Vortex vigorously until the sample has completely thawed and potential buffer precipitates have been dissolved again. Keep at room temperature and continue with further samples.
  10. Spin all samples 15 sec at 11,000 x g to get down the sample from the lid.
  11. Boil (95 °C) for 5-10 min.
  12. Let cool for 3 min.
  13. Centrifuge at 11,000 x g for 5 min to precipitate glass beads and debris.
  14. Take supernatant from the top (do not disturb the pellet) and load 15 μl on an SDS-PAGE gel (10% or 12%, 1 mm thickness).
  15. Perform your standard Western Blotting method (for us wet and semi-dry worked, and similarly both Alkaline Phosphatase and Horseradish peroxidase worked). We use the primary antibody at 1:2,000 dilutions.

Notes

  1. The “extraction buffer” used in this protocol is a 6x loading buffer. Usually a buffer called “Lacus” (which is very complex) is used for the extraction of phosphorylated MAPKs but we observed, that it works well with just adding the undiluted 6x loading buffer. Since it contains high amounts of SDS do not keep the samples on ice after adding the buffer. Since the samples are very cold SDS might precipitate anyway (white precipitate). In that vortex the samples at room temperature until the SDS dissolved again.
  2. This MAPK antibody detects in principle all phosphorylated MAPKs so it might produce more bands than just the two of MPK3 and MPK6 (especially when using other plant material). If possible include mpk3 and mpk6 mutants in your analysis to clarify the origin of the bands you see.
  3. According to the manufacturer the used MAPK antibody detects single as well as dual phosphorylated MAPKs. To our knowledge you cannot discriminate between the two states. Keep that in mind when interpreting the data.

Recipes

  1. 6x Protein extraction/loading buffer
    0.35 M Tris-HCl pH 6.8
    30% (v/v) glycerol
    10% (v/v) SDS
    0.6 M DTT
    0.012% (w/v) bromphenol blue

Acknowledgments

This work was supported by the Swiss National Science Foundation (grant 31003A_127563; to TB) and by stipends to SB from the European Molecular Biology Organisation (EMBO: ALTF 61-2010) and the Leopoldina Fellowship Programme of the National Academy of Science Leopoldina (LPDS 2009-35).

References

  1. Flury, P., Klauser, D., Schulze, B., Boller, T. and Bartels, S. (2013). The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst. Plant Physiol 161(4): 2023-2035.

简介

促分裂原活化蛋白激酶(MAPK)的激活涉及许多非生物和生物胁迫反应,包括植物防御。 MAPK激活基于MAPK蛋白激活环中苏氨酸(T)和酪氨酸(Y)残基(T-x-Y基序)的双重磷酸化。 通过确定特异性MAPK的磷酸化状态,可以检测MAPK是否已经被激活。
此协议描述如何使用叶片,western印迹和特异性抗体分析拟南芥MAPKs MPK3和MPK6的磷酸化状态(图1)。 它也可以用于分析其他植物系统中的MAPK,虽然可能需要关于蛋白质提取的一些改变。

关键字:拟南芥, 激酶, PTI, 先天免疫, 微生物



图1.用细菌激发剂鞭毛蛋白(+)的活性表位(flg22,1μM)处理幼苗15分钟时,检测拟南芥MAPKs MPK6和MPK3的磷酸化。 在对照处理的样品( - )中未检测到磷酸化MAPK。

材料和试剂

  1. 拟南芥成虫植物
  2. 液氮
  3. 磷酸-p44/42MAPK(Erk1/2)(Thr202/Tyr204)兔mAb#4370(Cell Signaling Technology)( http://www.cellsignal.com/products/4370.html
  4. 抗兔二抗
  5. Tris-HCl
  6. 甘油
  7. SDS
  8. 二硫苏糖醇(DTT)
  9. 溴酚蓝
  10. SDS-PAGE和Western印迹试剂
  11. 6x蛋白提取/加载缓冲液(参见配方)

设备

  1. 1.5 ml微量离心管
  2. 小培养皿(〜5cm直径)或6孔板
  3. 软木钻孔器(〜10 mm直径)
  4. 玻璃珠(〜1-2 mm直径)
  5. Silamat S6( http://www.ivoclarvivadent.com/en/products/设备/搅拌机/silamat-s6
  6. 或者为4.和5.砂浆(〜5厘米直径)和杵
  7. Vortexer
  8. 加热块
  9. 离心机
  10. SDS-PAGE,Western印迹和检测设备

程序

  1. 使用软木钻孔器从成年拟南芥(拟南芥)叶子切下叶盘。 尝试采取相似年龄和健身的叶子,但从不同的植物
  2. 称量一个示例性叶盘并记录重量(通常〜20mg)
  3. 在小培养皿或6孔板(每个皿至少3个或大约5ml水中的孔)中的蒸馏水上漂浮盘。
  4. 等待过夜(通常16小时,关闭盘子),并将盘子保持在不受干扰和受控的地方(例如原始生长室)。 这将导致在收获时被磷酸化的MAPK的去磷酸化
  5. 第二天早上进行治疗,例如添加一个你想知道如果它触发MAPK磷酸化的激发剂肽。我们将其添加到1μM的最终浓度,小心混合以不机械压迫叶组织,并且在大多数情况下等待15分钟。作为适当的对照,使用模拟处理(例如仅溶剂),并孵育15分钟。这是至关重要的,因为MAPK磷酸化很容易被许多不同的压力触发。因此,您需要证明您的对照治疗不会导致MAPK的磷酸化。其他治疗可以是添加化学物质,伤口紧张,紫外线治疗等。 注意:MAPK的磷酸化和激活发生非常快并且是短暂的。治疗后大约2-5分钟开始,持续至少1小时或更长时间。
  6. 在所需的孵育时间后,用镊子快速收获来自一个孔的所有盘,并在纸巾上短暂干燥(残留的水将稀释样品并干扰研磨程序)。将它们放入含有5个玻璃珠的微量离心管(优选安全锁)中,并在液氮中将它们骤冷。对每个孔重复此过程。
    注意:这里你必须快!收获盘(机械应力)将诱导MAPK磷酸化,因此,每个样品不应超过1分钟,以避免假阳性磷酸化结果!
  7. 研磨组织细粉(组织不应该解冻前下一步!)。你可以使用研钵和杵(在液氮中冷却),但我们不推荐这样做。特别是如果你有很多样品使用Silamat S6(或类似的机器)研磨组织,由于快速移动和先前添加的玻璃珠,研磨管中的组织。
  8. 添加提取/加载缓冲区。例如对于60毫克的组织,使用60微升,并根据样品重量使用相同的比例
  9. 剧烈涡旋直到样品完全解冻并且潜在的缓冲液沉淀物再次溶解。 保持室温,继续进一步的样品
  10. 旋转所有样品15秒,以11,000×g 从盖子上取下样品。
  11. 煮沸(95℃)5-10分钟
  12. 让冷静3分钟。
  13. 以11,000×g离心5分钟以沉淀玻璃珠和碎片
  14. 从顶部取上清(不要打扰沉淀),加入15μlSDS-PAGE凝胶(10%或12%,1mm厚)。
  15. 执行您的标准的Western印迹方法(对于我们湿和半干工作,并且类似地碱性磷酸酶和辣根过氧化物酶工作)。我们使用1:2,000稀释的一抗。

笔记

  1. 在该方案中使用的"提取缓冲液"是6x上样缓冲液。通常,称为"Lacus"(其非常复杂)的缓冲液用于提取磷酸化MAPK,但是我们观察到,仅通过添加未稀释的6x上样缓冲液就可以很好地工作。由于其含有大量的SDS,在加入缓冲液后不将样品保持在冰上。由于样品非常冷,SDS可能会沉淀(白色沉淀)。在该涡流中,样品在室温下,直到SDS再次溶解
  2. 这种MAPK抗体原则上检测所有磷酸化MAPK,因此其可以产生比仅MPK3和MPK6两者更多的条带(特别是当使用其他植物材料时)。如果可能,请在分析中包含 mpk3 和 mpk6 突变体,以澄清您看到的乐队的起源。
  3. 根据制造商,使用的MAPK抗体检测单个以及双重磷酸化MAPK。据我们所知,你不能区分这两种状态。在解释数据时请记住这一点。

食谱

  1. 6x蛋白提取/加载缓冲液
    0.35M Tris-HCl pH 6.8
    30%(v/v)甘油 10%(v/v)SDS
    0.6 M DTT
    0.012%(w/v)溴酚蓝

致谢

这项工作得到瑞士国家科学基金会(授予31003A_127563;结核病)和欧洲分子生物学组织(EMBO:ALTF 61-2010)和国家科学院Leopoldina Leopoldina奖学金计划(LPDS 2009-35)。

参考文献

  1. Flury,P.,Klauser,D.,Schulze,B.,Boller,T.and Bartels,S.(2013)。 预期危险:微生物相关分子模式感知增强AtPep触发的氧化爆发。 Plant Physiol 161(4):2023-2035。
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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。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Flury, P., Klauser, D., Boller, T. and Bartels, S. (2013). MAPK Phosphorylation Assay with Leaf Disks of Arabidopsis. Bio-protocol 3(19): e929. DOI: 10.21769/BioProtoc.929.
  2. Flury, P., Klauser, D., Schulze, B., Boller, T. and Bartels, S. (2013). The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst. Plant Physiol 161(4): 2023-2035.
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