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High-throughput Quantification of Ammonium Content in Arabidopsis
拟南芥铵含量的高通量测定   

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Abstract

This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction):

NH4+ + hypochlorite + OH- + phenol → indophenol

The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although it is also valid for other plants species.

Keywords: Ammonium(铵), Indophenol blue(靛酚蓝), Spectrophotometry(分光光度法), Plant(植物)

Materials and Reagents

  1. 4-weak old A. thaliana leaves and roots
  2. Ultrapure water (MilliQ) (EMD Millipore)
  3. Ice
  4. Liquid N2
  5. 4 mm diameter glass beads (Glaswarenfabrik Karl Hecht) (VWR International, catalog number: 201-0278 )
  6. EVA Capband for capping 8 tubes (Micronic, catalog number: MP227B1 )
  7. Flat bottom spectrophotometer microplates (Deltalab, catalog number: 900011.1 )
  8. 1.2 ml 96-well storage plate (Thermo Fisher Scientific, catalog number: AB-0564 )
  9. Sodium phenolate or sodium phenoxide trihydrate (Sigma-Aldrich, catalog number: 318191 )
  10. Sodium nitroprusside dihydrate (Sigma-Aldrich, catalog number: s0501 )
  11. Commercial bleach or Sodium Hypochlorite solution 10% (Panreac Applichem, catalog number: 211921 )
  12. Solution A (see Recipes)
  13. Solution B (see Recipes)
  14. Solution C (see Recipes)
  15. 10 mM NH4+ stock (for standard curve) (see Recipes)

Equipment

  1. TissueLyser (Retsch, model: MM400 )
  2. TissueLyser Adapter Set 2 x 96 (QIAGEN, catalog number: 69984 )
  3. Plate centrifuge (Sigma, model: 2-16K )
  4. Drying oven
  5. Absorbance microplate reader (Biotek PowerWave X 340 Microplate Spectrophometer)

Procedure

  1. Standard curve
    1. Prepare dilutions from the 10 mM NH4+ stock in a 0.05 mM - 1 mM range and use ultrapure water as blank. For example: blank; 0.05 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM.

  2. Material harvest and homogenization
    1. In a 96 deepwell (1.2 ml) plate, place 2 glass beads (4 mm diameter) into each well.
    2. Weight approximately 20 mg of fresh leaves or roots and place them in a microplate deepwell half-submerged in liquid nitrogen. Store at -80 °C until use.
    3. Grind frozen tissue with a TissueLyser two times for 60 sec at 27 Hz frequency. The plates are coupled to the TissueLyser with the TissueLyser Adapter Set 2 x 96. (Ensure sealing of the wells during homogenization).

  3. Extraction
    1. Add 500 μl ultrapure water per well.
    2. Grind again as in step B3 (make sure the water is not frozen before grinding).
    3. Incubate samples at 80 °C in a drying oven for 10 min (the incubation can be also done in a water bath).
    4. Centrifuge the plate at 4,000 rpm and 4 °C for 20 min in a plate centrifuge.
    5. Recover supernatants (400 μl) in a new 96 deepwell plate and keep the samples on ice.

  4. Ammonium content measurement
    1. Place 50 µl of extract (or 50 µl of standards for the calibration curve) per well, in a 96 well spectrophotometer microplate.
    2. Add 100 µl of Solution A, 50 µl of Solution B and finally 100 µl of Solution C to every sample.
    3. Make blank using 50 µl of water instead of the plant extract.
    4. Incubate 30 min at room temperature.
    5. Read absorbance at 635 nm in a spectrophotometer (Figure 1).
    6. Making three technical replicates per sample or standard is recommended.


    Figure 1. Representative ammonium standard curve. When ammonium content is higher than 1 nmol NH4+ per microliter of reaction mixture absorbance starts to get saturated. Linear regression is performed before arriving to saturation.

Data analysis

Subtract the blank absorbance from the absorbance of every sample and calculate the ammonium concentration with the standards calibration curve. Finally, express the ammonium content in a fresh weight basis. These calculations might be also done directly by the microplate reader software assigning to every well a category for example, blank, standard or sample.

Notes


  1. This protocol can be easily adapted to Eppendorf tubes and spectrophotometer cuvettes instead of 96-well plates just scaling the volumes. Similarly, the homogenisation of the samples might be done with a mortar and pestle instead of the TissueLyser.
  2. If possible, we advice to do every 96-well plate step using multi-pipettes to save time and thus to gain reproducibility among samples.


Recipes

  1. Solution A
    0.33 M sodium phenolate
    Prepare 2 M NaOH dissolving 8 g NaOH in 100 ml of ultrapure water
    Mix 2.8 g of sodium phenolate with 4 ml of 2 M NaOH in a final volume of 50 ml with ultrapure water
    Ensure pH of the solution is around 13
  2. Solution B
    0.02% sodium nitroprusside
    Dissolve 20 mg of sodium nitroprusside in 100 ml of water
    Prepare just before using
  3. Solution C
    2% sodium hypochlorite
    Dilute 1 ml of commercial bleach in 49 ml of ultrapure water
  4. 10 mM NH4+ stock (for standard curve)
    Dissolve 33.025 mg of (NH4)2SO4 in 50 ml of ultrapure water (of course NH4Cl can also be used as standard)

Acknowledgments

This protocol is adapted from Sarasketa et al. (2014) and based on the methodology reported by Solorzano (1969). This work was supported by the Basque Government (IT526-10), the UPV/EHU (EHUA14/14), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA grant agreement number 334019 and MINECO (BIO2014-56271-R).

References

  1. Sarasketa, A., Gonzalez-Moro, M. B., Gonzalez-Murua, C. and Marino, D. (2014). Exploring ammonium tolerance in a large panel of Arabidopsis thaliana natural accessions. J Exp Bot 65(20): 6023-6033.
  2. Solorzano L. (1969). Determination of ammonium in natural waters by the phenol-hypochlorite method. Limnol Oceanogr 14: 799-801.

简介

该方案是一种简单的比色测定法,用于在植物组织的水性提取物中进行内部铵定量。 该方法基于苯酚次氯酸盐测定(Berthelot反应):
NH 4+ sup + +次氯酸盐+ OH - +苯酚→靛酚

由酚氧化引起的靛酚的氧化是在分光光度计中在635nm处定量的蓝色染料。 每个铵分子形成一个靛酚分子。 这里描述的方案是用于拟南芥(拟南芥)叶和根,虽然它也适用于其他植物物种。

关键字:铵, 靛酚蓝, 分光光度法, 植物

材料和试剂

  1. 4弱老 A。 thaliana 叶和根
  2. 超纯水(MilliQ)(EMD Millipore)

  3. 液体N <2>
  4. 4mm直径的玻璃珠(Glaswarenfabrik Karl Hecht)(VWR International,目录号:201-0278)
  5. 用于封盖8根管的EVA帽带(Micronic,目录号:MP227B1)
  6. 平底分光光度计微量培养板(Deltalab,目录号:900011.1)
  7. 1.2ml 96孔培养板(Thermo Fisher Scientific,目录号:AB-0564)
  8. 苯酚钠或三水合苯酚钠(Sigma-Aldrich,目录号:318191)
  9. 硝普钠二水合物(Sigma-Aldrich,目录号:s0501)
  10. 商业漂白剂或次氯酸钠溶液10%(Panreac Applichem,目录号:211921)
  11. 解决方案A(参见配方)
  12. 解决方案B(参见配方)
  13. 解决方案C(参见配方)
  14. 10mM NH 4 + 储液(对于标准曲线)(参见配方)

设备

  1. TissueLyser(Retsch,型号:MM400)
  2. 解决方案A(参见配方)
  3. 解决方案B(参见配方)
  4. 解决方案C(参见配方)
  5. 10mM NH 4 + 储液(对于标准曲线)(参见配方)

设备

  1. TissueLyser(Retsch,型号:MM400)
  2. ...
    1. Prepare dilutions from the 10 mM NH4+ stock in a 0.05 mM - 1 mM range and use ultrapure water as blank. For example: blank; 0.05 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM.

  3. Material harvest and homogenization
    1. In a 96 deepwell (1.2 ml) plate, place 2 glass beads (4 mm diameter) into each well.
    2. Weight approximately 20 mg of fresh leaves or roots and place them in a microplate deepwell half-submerged in liquid nitrogen. Store at -80 °C until use.
    3. Grind frozen tissue with a TissueLyser two times for 60 sec at 27 Hz frequency. The plates are coupled to the TissueLyser with the TissueLyser Adapter Set 2 x 96. (Ensure sealing of the wells during homogenization).

  4. Extraction
    1. Add 500 μl ultrapure water per well.
    2. Grind again as in step B3 (make sure the water is not frozen before grinding).
    3. 在80℃的干燥箱中孵育样品10分钟(孵育也可以在水浴中进行)。
    4. 在板式离心机中以4,000rpm和4℃离心板20分钟
    5. 回收在新的96深孔板中的上清液(400μl),并保持在冰上的样品。

  5. 铵含量测量
    1. 放置50μl的提取物(或50μl的标准品的校准 曲线),在96孔分光光度计微孔板中。
    2. 向每个样品中加入100μl溶液A,50μl溶液B和最后100μl溶液C.
    3. 使用50微升水代替植物提取物,使空白
    4. 在室温下孵育30分钟。
    5. 在分光光度计(图1)中读取635 nm处的吸光度。
    6. 建议每个样品或标准进行三次技术重复。


    图1.代表性铵标准曲线当铵含量高于每分钟1微升反应混合物的1nmol NH 4+时,吸收开始饱和 。 线性回归是 在达到饱和之前执行

数据分析

从每个样品的吸光度减去空白吸光度,并用标准品校准曲线计算铵浓度。 最后,以鲜重表示铵含量。 这些计算也可以通过微孔板读数器软件直接进行,分配给每个孔一个类别,例如空白,标准或样品。

笔记


  1. 这个协议可以很容易地适应于Eppendorf管和 分光光度计比色皿代替96孔板只是缩放 卷。 类似地,样品的均质化可以用   研钵和杵替代TissueLyser。
  2. 如果可能,我们建议做每一个96孔板步骤使用 多移液器以节省时间,从而获得重现性 样品。


食谱

  1. 解决方案A
    0.33M苯酚钠
    准备2 M NaOH溶解8 g NaOH在100 ml超纯水中
    用超纯水将2.8g苯酚钠与4ml 2M NaOH混合,最终体积为50ml 确保溶液的pH值约为13
  2. 解决方案B
    0.02%硝普钠
    将20mg硝普钠溶于100ml水中
    在使用
    前准备
  3. 解决方案C
    2%次氯酸钠
    将1ml商业漂白剂稀释在49ml超纯水中
  4. 10mM NH 4 + 储液(对于标准曲线)
    将33.025mg的(NH 4)2 SO 4溶解在50ml超纯水(当然NH 4)中, Cl也可以作为标准)

致谢

该协议根据Solorzano(1969)报道的方法从Sarasketa等人(2014)修改。这项工作得到了欧盟第七框架计划(FP7/2007-2013)的巴斯克政府(IT526-10),UPV/EHU(EHUA14/14),人民计划(Marie Curie Actions)编号334019和MINECO(BIO2014-56271-R)。

参考文献

  1. Sarasketa,A.,Gonzalez-Moro,M.B.,Gonzalez-Murua,C.and Marino,D。(2014)。 在大面板的拟南芥自然种质中探索铵耐受性。/a> J Exp Bot 65(20):6023-6033
  2. Solorzano L.(1969)。 通过酚 - 次氯酸盐方法测定天然水中的铵。 Limnol Oceanogr 14:799-801 />
  • 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: © 2015 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. Vega-Mas, I., Sarasketa, A. and Marino, D. (2015). High-throughput Quantification of Ammonium Content in Arabidopsis. Bio-protocol 5(16): e1559. DOI: 10.21769/BioProtoc.1559.
  2. McAdam, S. A. and Brodribb, T. J. (2014). Separatingactive and passive influences on stomatal control of transpiration. Plant Physiol 164(4): 1578-1586.
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