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Assay of Arabinofuranosidase Activity in Maize Roots
玉米根中阿拉伯呋喃糖酶的活性测定   

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Abstract

Root is a perfect model for studying the mechanisms of plant cell growth. Along the root length, several zones where cells are at different stages of development can be visualized (Figure 1). The dissection of the root on these zones allows the investigation of biochemical and genetic aspects of different growth steps. Maize primary root is much more massive than the root of other Monocots and thus more convenient for such type of research. Plant cell wall, mainly consisting of polysaccharides, plays an important role in plant life. Therefore, measurement of plant carbohydrate content and glycoside-modifying enzyme activity in plant cells has become an important aspect in plant physiology. One of the well-documented changes of hemicelluloses molecules during elongation growth of monocots cells is the decrease of arabinose substitution of glucuronoarabinoxylans. This might be caused by changes in synthesis of this polysaccharide or by the action of arabinofuranosidases. Here, we describe the protocol of spectrophotometric measuring of arabinofuranosidase activity in maize root by the rate of hydrolysis of chromogenic substrate (4-nitrophenyl α-L-arabinofuranoside).


Figure 1. Scheme of plant material collection for further arabinofuranosidase assay. Four-day-old dark-grown maize seedling (left panel). Different zones of primary maize root and corresponding stages of cell development, according to Kozlova et al. (2012) (right panel).

Materials and Reagents

  1. Microcentrifuge tubes 1.5-2 ml
  2. CarboPac PA1 analytical column (4 x 250 mm) (Thermo Fisher Scientific, catalog number: 035391 )
  3. CarboPac PA1 guard column (4 x 50 mm) (Thermo Fisher Scientific, catalog number: 043096 )
  4. Maize seedlings
  5. 4-nitrophenyl α-L-arabinofuranoside (4NPA) (Sigma-Aldrich, catalog number: N3641 )
  6. 4-nitrophenol (NP) (spectrophotometric grade) (Sigma-Aldrich, catalog number: 1048 )
  7. DL-Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
  8. Quick StartTM Bradford 1x Dye (Bio-Rad Laboratories, AbD Serotec®, catalog number: 5000205 )
  9. Sodium Acetate Anhydrous (NaOAc) (Sigma-Aldrich, catalog number: W302406 )
  10. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 306576 )
  11. L-(+)-ARABINOSE (Sigma-Aldrich, catalog number: W325501 )
  12. NaN3
  13. Na2CO3
  14. NaOCl
  15. Glacial acetic acid
  16. Ice
  17. MilliQ water (18 MΩ-cm)
  18. 5% (w/w) solution of NaOCl (see Recipes)
  19. 0.03% (w/w) NaN3 (see Recipes)
  20. 50 mM NaOAc buffer (pH 6.0) with 3 mM of dithiothreitol on 0.03% (w/w) NaN3 (see Recipes)
  21. 10 mM 4NPA (see Recipes)
  22. 1 M Na2CO3 (see Recipes)
  23. Calibration solutions for NP (see Recipes)
  24. 50% (w/w) NaOH solution (see Recipes)
  25. 15 mM NaOH (buffer A for HPAEC) (see Recipes)
  26. 1 M NaOAc in 0.1 M NaOH (buffer B for HPAEC) (see Recipes)
  27. Calibration solutions for arabinose (see Recipes)

Equipment

  1. Spectrophotometer (required wavelength is 405 nm)
  2. Thermostat (required temperature is 27 °C)
  3. Microcentrifuge (required spin rate is 10,000 x g)
  4. Thermoshaker (required temperature and spin rate are 37 °C and 600 rpm, correspondingly)
    Note: Thermoshaker should be appropriate for incubation of microcentrifuge tubes.
  5. Mini centrifuge/vortex
  6. Analytical balance with 0.1 mg readability
  7. Ion chromatography system DX-500 (Thermo Fisher Scientific, Dionex) equipped by electrochemical detector ED40, gradient pump GP40 and chromatography oven LC 30
  8. Refrigerator (required temperature is -20 °C)
  9. Automatic pipettes (100-1,000, 20-200 and 2-20 μl)
  10. Mortar and pestle
  11. Plastic grid and the tray of proper size
    Note: The cell of grid should be approximately 1 cm2 or slightly bigger.

Software

  1. PeakNet software (Thermo Fisher Scientific, Dionex, model: version 5.21)

Procedure

Principal scheme of the experiment is shown in Figure 2.


Figure 2. Scheme of the experiment

  1. Seeds of maize (Zea mays L.) were sterilized in 5% (w/w) solution of NaOCl for 10 min then were washed twice in water. Seeds were placed in the cells of plastic grid that was mounted in a tray with water. Plants were grown in the dark in a thermostat at 27 °C for 96 h. While any part of etiolated seedling can be used for the assay, we have used five zones (meristem, early elongation, elongation, late elongation, post-elongation) of primary roots (Figure 1). The recommended amount of plant material in each sample is 30-40 mg. One mm of maize root represents approximately one mg of fresh weight. Thus, root pieces of 10 mm length from three or four seedlings or pieces of 1-mm length from 30-40 seedlings are enough for one replica.
  2. 30-40 mg of fresh plant material was homogenized with mortar and pestle on ice in 1 ml of 50 mM NaOAc buffer (pH 6.0) with 3 mM of dithiothreitol to prevent the intramolecular and intermolecular disulfide bonds formation and with 0.03% (w/w) NaN3 to restrict bacterial contamination. Homogenate was transferred to microcentrifuge tube (1.5-2 ml). Homogenates that were already prepared could be stored in refrigerator (-20 °C) while other samples are being done.
  3. 1 ml of suspension was centrifuged for 10 min at 10,000 x g. Then 100 μl of supernatant were separated for further estimation of protein content according to Bradford (1976) (step 9) and free arabinose content estimation (step 10). Supernatants could be stored in refrigerator (-20 °C). Steps 9-10 could be carried out later. The arabinose and protein contents do not change if samples are frozen.
  4. Pellet was resuspended in the remaining 900 μl of buffer solution using Vortex. Plant material was not separated from supernatant because of the potential presence of wall-associated arabinofuranosidases that might be not extracted by the buffer solution. The suspension was divided into two equal parts (450 μl each) that were transferred to plastic tubes as control and trial variants of each sample.
  5. 50 μl of 10 mM 4NPA in 0.03% (w/w) NaN3 was added to the trial tubes, and 50 μl of 0.03% (w/w) NaN3 to the control tubes. Both of the variants were incubated in the thermoshaker at 600 rpm at 37 °C for 2 h. During that time cell wall arabinofuranosidases (either soluble or wall-associated) in trial tubes removed arabinose residues from 4NPA molecules producing yellow-colored NP. Enzymes in control tubes had no chromogenic substrate for reaction.
  6. The reaction was stopped by the addition of 500 μl of 1 M Na2CO3 both to the control and to the trial tubes. The coloration of the solution has become more pronounced at that moment due to increased pH (11.5).
  7. Samples were centrifuged for 10 min at 10,000 x g. Optical densities of both trial and control supernatants were measured on a spectrophotometer at wavelength 405 nm. The control mean was subtracted from the trial mean. The result was used for the determination of the amount of liberated NP according to calibration.
  8. Calibration measurements were done for NP solutions with concentrations in a range from 0.0025 mM to 0.25 mM (Figure 3). 500 μl of 1 M Na2CO3 were added to 500 μl of each NP solution, because it enhances the coloration.
  9. Determination of protein content.
    1. 490 μl of water was added to 10 μl aliquots of supernatant from step 3 of each sample. 500 μl of Bradford dye reagent was also added to each microcentrifuge tube and then mixed using vortex.
    2. After 5 min each sample was read on a spectrophotometer at 595 nm.
  10. Determination of free arabinose content.
    1. 90 μl of water waw added to 10 μl aliquots of supernatant from step 3 of each sample.
    2. 25 μl of obtained solution was directly injected to the column of HPAEC system without any pretreatment.
    3. 15 mM NaOH (buffer A) and 1 M NaOAc in 0.1 M NaOH (buffer B) were used as eluents. The column temperature was maintained at 30 °C, and the elution rate was 1 ml/min. The gradient elution was performed as follows: 0-20 min-100% A; 20-21 min-linear gradient until the A/B ratio = 90:10(%); 21-31 min-linear gradient until the A/B ratio = 70:30(%); 31-32 min-linear gradient until the A/B ratio 0:100(%); 32-42 min-100% B; 42-43 min-until the A/B ratio = 100:0(%). Column was equilibrated with eluent 100% A for 30 min.
    4. Arabinose content was determined by the area of corresponding peak using the PeakNet software and the calibration for arabinose standard.
  11. Calibration for free arabinose was done using solutions with concentrations in a range 2-10 μg/ml.


    Figure 3. Calibration for different nitrophenol (NP) concentrations. Do not confuse the concentration and the amount of NP matter in reaction volume (see Table 1).

Notes

  1. Experiments should be repeated three-four times for each sample.
  2. One unit of arabinofuranosidase activity corresponds to the amount of enzyme that liberates one μmol of NP per minute. Differences of glycosyl-hydrolase activities between samples can be defined by using the units of enzymatic activity per mg of total proteins. Exact formula for arabinofuranosidase activity calculation is given in Figure 4.


    Figure 4. Formula for calculation of arabinofuranosidases activity. The result will be obtained in milliUnits of activity per mg of protein content.

  3. Described protocol with small modifications can be used for determination of any other glycosyl-hydrolase activity with synthetic aryl glycosides. Preliminary experiments may be useful for the optimization of incubation time and of plant material amount.
    However, the enzyme activity that is measured only by the rate of chromogenic substrate hydrolysis can remain the potential one and can be not realized in plant tissues (Fry, 2004). The aryl-glycoside hydrolysis coupled with the demonstration of final product presence is much more convincing. Thus, steps 10 and 11 were included in protocol. If for any reason you cannot perform these stages, you may conclude only about potential activity of arabinofuranosidases.

Recipes

Note: The recipes that are given below let to produce the amount of solutions that is sufficient for calibration and four repeating of the experiments with five samples in each replica.

  1. 5% (w/w) solution of NaOCl
    Dissolve 25 g of NaOCl in 475 ml of water
  2. 0.03% (w/w) NaN3
    Dissolve 0.03 g of NaN3 in 99.97 ml of water
  3. 50 mM NaOAc buffer (pH 6.0) with 3 mM of dithiothreitol on 0.03% (w/w) NaN3
    Dissolve 0.205 g of NaOAc and 0.0231 g of DL-dithiothreitol in 40 ml of 0.03% (w/w) NaN3
    Adjust the pH to 6.0 by the adding of glacial acetic acid
    Adjust the volume to 50 ml by the adding of 0.03% (w/w) NaN3
  4. 10 mM 4NPA
    Dissolve 0.0027 g of 4NPA in 0.03% (w/w) NaN3
    Adjust the volume of solution to 1 ml by additional 0.03% (w/w) NaN3
  5. 1 M Na2CO3
    Dissolve 2.12 g of Na2CO3 in 0.03% (w/w) NaN3
    Adjust the volume of solution to 20 ml by additional 0.03% (w/w) NaN3
  6. Calibration solutions for NP
    Dissolve 0.0139 g of NP in 1 ml of 0.03% (w/w) NaN3 to produce 100 mM NP solution
    Add 10 μl of 100 mM NP solution to 990 μl of 0.03% (w/w) NaN3 to produce 1 mM NP solution
    Prepare 9 concentrations of NP according to the table below, place 500 μl of each calibration solution to individual tubes and add 500 μl of 1M Na2CO3 to all.

    Table 1. NP solution preparation
    NP concentration, mM
    Amount of NP matter in reaction volume (1 ml), μmol
    Volume of 1 mM NP solution, μl
    0.03% (w/w) NaN3 solution volume, μl
    0.25
    0.125
    250
    750
    0.1
    0.05
    100
    900
    0.05
    0.025
    50
    950
    0.025
    0.0125
    25
    975
    0.02
    0.01
    20
    980
    0.0125
    0.00625
    12.5
    987.5
    0.01
    0.005
    10
    990
    0.005
    0.0025
    5
    995
    0.0025
    0.00125
    2.5
    997.5

  7. 50% (w/w) NaOH solution
    HPAEC buffers should be prepared using template solution of NaOH
    Add 5 ml of degasified MilliQ water (18 MΩ-cm) to 5 g of NaOH
  8. 15 mM NaOH (buffer A for HPAEC)
    Add 780 μl of 50% NaOH in 1,000 ml of degasified MilliQ water (18 MΩ-cm)
  9. 1 M NaOAc in 0.1 M NaOH (buffer B for HPAEC)
    Dissolve 82.05 g of NaOAc in degasified MilliQ water (18 MΩ-cm)
    Adjust volume of solution to 1,000 ml by the adding of degasified MilliQ water (18 MΩ-cm)
    Add 5.23 ml of 50% NaOHto NaOAc solution
  10. Calibration solutions for arabinose
    Dissolve 0.001 g of arabinose in 1 ml of MilliQ water to produce 10 mg/ml solution
    Add 100 μl of 10 mg/ml arabinose solution to 900 μl of MilliQ water to obtain 1 mg/ml solution
    Prepare 5 solutions of arabinose according to Table 2 below

    Table 2. Arabinose solution preparation
    Arabinose concentration, μg/ml
    Volume of 1 mg/ml arabinose solution, μl
    Volume of MilliQ, μl
    2
    2
    998
    3
    3
    997
    5
    5
    995
    8
    8
    992
    10
    10
    990

Acknowledgments

This protocol was adapted from the previously published study (Kozlova et al., 2015). This work was partially supported by Russian Foundation for Basic Research (project ## 14-04-01002 and 15-04-02560).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Fry, S. C. (2004). Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytol 161:641-675.
  3. Kozlova, L. V., Gorshkov, O. V., Mokshina, N. E. and Gorshkova, T. A. (2015). Differential expression of alpha-L-arabinofuranosidases during maize (Zea mays L.) root elongation. Planta 241(5): 1159-1172.
  4. Kozlova, L. V., Snegireva A. V. and Gorshkova T. A. (2012). Distribution and structure of mixed linkage glucan at different stages of elongation of maize root сells. Russ J Plant Phys 59: 339-347.

简介

根是研究植物细胞生长机制的完美模型。沿着根长度,可以可视化细胞处于不同发育阶段的几个区(图1)。在这些区域上的根的解剖允许调查不同生长步骤的生化和遗传方面。玉米主根比其他单根的根更大,因此这种类型的研究更方便。植物细胞壁,主要由多糖组成,在植物生活中发挥重要作用。因此,植物碳水化合物含量和糖苷修饰酶活性在植物细胞中的测量已经成为植物生理学中的重要方面。在单子叶植物细胞的延长生长期间半纤维素分子的充分证明的变化之一是葡糖醛酸阿拉伯木聚糖的阿拉伯糖取代的减少。这可能是由于该多糖的合成的变化或阿拉伯呋喃糖苷酶的作用引起的。在这里,我们描述的分光光度测量阿拉伯呋喃糖苷酶活性在玉米根中的水解发色底物(4-硝基苯基α-L-阿拉伯呋喃糖苷)的速率的协议。


图1. 四日龄黑暗生长的玉米幼苗面板)。根据Kozlova等人(2012)(右图),初级玉米根的不同区和细胞发育的相应阶段。

材料和试剂

  1. 微量离心管1.5-2 ml
  2. CarboPac PA1分析柱(4×250mm)(Thermo Fisher Scientific,目录号:035391)
  3. CarboPac PA1保护柱(4×50mm)(Thermo Fisher Scientific,目录号:043096)
  4. 玉米苗
  5. 4-硝基苯基α-L-阿拉伯呋喃糖苷(4NPA)(Sigma-Aldrich,目录号:N3641)
  6. 4-硝基苯酚(NP)(分光光度级)(Sigma-Aldrich,目录号:1048)
  7. DL-二硫苏糖醇(Sigma-Aldrich,目录号:D0632)
  8. 快速启动 Bradford 1x Dye(Bio-Rad Laboratories,AbD Serotec ,目录号:5000205)
  9. 无水乙酸钠(NaOAc)(Sigma-Aldrich,目录号:W302406)
  10. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:306576)
  11. L - (+) - 阿拉伯糖(Sigma-Aldrich,目录号:W325501)
  12. NaN 3
  13. Na 2 3
  14. NaOCl
  15. 冰醋酸

  16. MilliQ水(18MΩ-cm)
  17. 5%(w/w)NaOCl溶液(参见配方)
  18. 0.03%(w/w)NaN 3(参见配方)
  19. 50mM NaOAc缓冲液(pH6.0),其中在0.03%(w/w)NaN 3上具有3mM二硫苏糖醇(参见配方)
  20. 10 mM 4NPA(参见配方)

  21. (参见配方)。
  22. NP的校准解决方案(参见配方)
  23. 50%(w/w)NaOH溶液(参见配方)
  24. 15mM NaOH(HPAEC的缓冲液A)(参见Recipes)
  25. 1M NaOAc在0.1M NaOH(HPAEC的缓冲液B)(参见配方)中
  26. 阿拉伯糖的校准溶液(参见配方)

设备

  1. 分光光度计(所需波长为405nm)
  2. 恒温器(所需温度为27°C)
  3. 微量离心机(所需旋转速率为10,000×g )
  4. Thermoshaker(所需温度和旋转速率分别为37℃和600rpm)
    注意:Thermoshaker应适合孵育微量离心管。
  5. 迷你离心机/涡流
  6. 具有0.1 mg可读性的分析天平
  7. 装备有电化学检测器ED40,梯度泵GP40和色谱炉LC30的离子色谱系统DX-500(Thermo Fisher Scientific,Dionex)
  8. 冰箱(所需温度为-20°C)
  9. 自动移液器(100-1,000,20-200和2-20μl)
  10. 砂浆和杵
  11. 塑料网格和适当尺寸的托盘
    注意:网格单元格大约为1平方厘米或稍大。

软件

  1. PeakNet软件(Thermo Fisher Scientific,Dionex,型号:5.21)

程序

实验的主要方案如图2所示。


图2.实验方案

  1. 将玉米种子(Zea mays L.)在5%(w/w)的 NaOCl洗涤10分钟,然后在水中洗涤两次。将种子放入 塑料网格的细胞,其被安装在具有水的托盘中。植物 在黑暗中在27℃的恒温箱中生长96小时。而任何部分 的化学幼苗可用于测定,我们使用五个区 ?(分生组织,早期伸长,伸长,晚期伸长, 后伸长)(图1)。推荐量 每个样品中的植物材料为30-40mg。 1毫米的玉米根 代表约1mg鲜重。因此,根块10 ?mm长度从三个或四个幼苗或1毫米长度的片段 30-40棵幼苗足够一次复制
  2. 30-40毫克新鲜 植物材料用研钵和杵在冰上在1ml的水中均化 ?50mM NaOAc缓冲液(pH6.0)与3mM二硫苏糖醇混合以防止 分子内和分子间二硫键形成和与 0.03%(w/w)NaN 3以限制细菌污染。均匀 转移至微量离心管(1.5-2ml)。均匀 已经准备好可以存储在冰箱(-20°C),而其他 正在处理样本。
  3. 将1ml悬浮液离心10分钟 ?min,10,000×g/g。然后分离100μl上清液 根据Bradford(1976)进一步估计蛋白质含量(步骤 ?9)和游离阿拉伯糖含量估计(步骤10)。上清可以 储存在冰箱(-20°C)中。可以执行步骤9-10 后来。如果样品是阿拉伯糖和蛋白质含量不变 冻结。
  4. 将沉淀重悬于剩余的900μl缓冲液中 ?解决方案。植物材料未从中分离 上清液由于可能存在与壁相关 可能不能被缓冲溶液萃取的阿拉伯呋喃糖苷酶。 ?将悬浮液分成两等份(每份450μl) 转移到塑料管作为对照和每个的试验变体 样品
  5. 将50μl在0.03%(w/w)NaN 3中的10mM 4NPA加入到 试管中,并向对照管中加入50μl的0.03%(w/w)NaN 3。两者的 ?将变体在37℃下在600rpm下在热水瓶中温育 ?2小时。在此期间细胞壁阿拉伯呋喃糖苷酶(可溶性或 ?壁相关)在试管中从4NPA中移除阿拉伯糖残基 分子产生黄色NP。对照管中的酶没有 显色底物反应
  6. 通过反应停止反应 向对照和向对照中加入500μl的1M Na 2 CO 3 SO 3。 试管。溶液的着色变得更加显着 此时由于pH增加(11.5)
  7. 样品 在10,000×g离心10分钟。两种试验的光密度 并在波上在分光光度计上测量对照上清液 长度405nm。从试验平均值中减去对照平均值。的 结果用于测定释放的NP的量 根据校准。
  8. 进行校准测量 NP溶液,其浓度在0.0025mM至0.25mM的范围内 (图3)。将500μl的1M Na 2 CO 3 3加入到500μl的每种NP中 解决方案,因为它增强了着色
  9. 测定 蛋白质含量。
    1. 将490μl水加入10μl等分试样中 来自每个样品的步骤3的上清液。 500μl的Bradford染料试剂 也加入每个微量离心管中,然后使用混合 涡流。
    2. 5分钟后,每个样品在分光光度计上在595nm读数。
  10. 游离阿拉伯糖含量的测定。
    1. 加入90μl水 至10μl来自每个样品的步骤3的上清液的等分试样。
    2. 25μl 得到的溶液直接注入HPAEC系统柱 无需任何预处理。
    3. 15mM NaOH(缓冲液A)和1M NaOAc的0.1M溶液 NaOH(缓冲液B)用作洗脱剂。柱温度 保持在30℃,洗脱速率为1ml/min。梯度 洗脱如下进行:0-20分钟-100%A; 20-21分钟线性 梯度直到A/B比= 90:10(%); 21-31分钟线性梯度 ?A/B比= 70:30(%); 31-32分钟 - 线性梯度直到A/B比 ?0:100(%); 32-42分钟-100%B; 42-43分钟,直到A/B比= 100:0(%)。 将柱用100%A的洗脱液平衡30分钟。
    4. 阿拉伯糖含量 ?通过使用PeakNet的相应峰的面积确定 软件和阿拉伯糖标准的校准。
  11. 使用浓度范围为2-10μg/ml的溶液进行游离阿拉伯糖的校准

    图3.不同硝基苯(NP)浓度的校准。 ?不混淆反应中NP物质的浓度和量 体积(见表1)。

笔记

  1. 对于每个样品,实验应重复三到四次。
  2. 一个单位的阿拉伯呋喃糖苷酶活性对应于每分钟释放1μmolNP的酶量。样品之间的糖基水解酶活性的差异可以通过使用每mg总蛋白的酶活性单位来定义。阿拉伯呋喃糖苷酶活性计算的精确公式在图4中给出

    图4.计算阿拉伯呋喃糖苷酶活性的公式。结果将以mg蛋白质含量的活性单位获得。

  3. 具有小修饰的描述方案可用于用合成的芳基糖苷测定任何其它糖基水解酶活性。初步实验可用于优化培养时间和植物材料量 然而,仅通过发色底物水解的速率测量的酶活性可以保持潜在的并且可以在植物组织中不实现(Fry,2004)。芳基 - 糖苷水解与最终产物存在的证明相结合是更有说服力的。因此,步骤10和11包括在方案中。如果由于任何原因,您无法执行这些阶段,您可能只得出阿拉伯呋喃糖苷酶的潜在活性。

食谱

注意:下面给出的配方允许产生足以进行校准的溶液的量,以及四次重复的实验,每个复制品中有五个样品。

  1. 5%(w/w)的NaOCl溶液 将25g NaOCl溶于475ml水中
  2. 0.03%(w/w)NaN 3·
    将0.03g NaN 3溶于99.97ml水中
  3. 50mM NaOAc缓冲液(pH 6.0)中用3mM二硫苏糖醇在0.03%(w/w)NaN 3缓冲液 将0.205g NaOAc和0.0231g DL-二硫苏糖醇溶解在40ml的0.03%(w/w)NaN 3中。 通过加入冰醋酸将pH调节至6.0 通过加入0.03%(w/w)NaN 3
    将体积调节至50ml
  4. 10 mM 4NPA
    将0.0027g的4NPA溶解在0.03%(w/w)NaN 3中。
    通过额外的0.03%(w/w)NaN 3/v/v将溶液的体积调节至1ml
  5. 1 M Na 2 CO 3 sub
    在0.03%(w/w)NaN 3中溶解2.12g Na 2 CO 3。 通过额外的0.03%(w/w)NaN 3/v/v将溶液的体积调节至20ml。
  6. NP的校准解决方案
    将0.0139g NP溶解在1ml的0.03%(w/w)NaN 3中以产生100mM NP溶液
    将10μl的100mM NP溶液加入到990μl的0.03%(w/w)NaN 3中以产生1mM NP溶液。
    根据下表准备9个NP浓度,放置500μl 的每个校准溶液加入到单独的管中并加入500μl的1M Na 2 CO 3
    表1. NP解决方案准备
    NP浓度,mM
    反应中的NP物质的量 体积(1ml),μmol
    1mM NP溶液体积,μl
    0.03%(w/w)NaN 3溶液体积,μl
    0.25
    0.125
    250
    750
    0.1
    0.05
    100
    900
    0.05
    0.025
    50
    950
    0.025
    0.0125
    25
    975
    0.02
    0.01
    20
    980
    0.0125
    0.00625
    12.5
    987.5
    0.01
    0.005
    10
    990
    0.005
    0.0025
    5
    995
    0.0025
    0.00125
    2.5
    997.5

  7. 50%(w/w)NaOH溶液 HPAEC缓冲液应使用NaOH模板溶液制备 向5g NaOH中加入5ml去气的MilliQ水(18MΩ-cm)
  8. 15mM NaOH(HPAEC的缓冲液A)
    在1000ml脱气的MilliQ水(18MΩ-cm)中加入780μl的50%NaOH
  9. 1M NaOAc的0.1M NaOH溶液(HPAEC的缓冲液B)
    将82.05g NaOAc溶解在脱气的MilliQ水(18MΩ-cm)中 通过加入脱气的MilliQ水(18MΩ-cm)将溶液的体积调节至1,000ml 加入5.23ml 50%NaOH至NaOAc溶液
  10. 阿拉伯糖的校准溶液
    将0.001g阿拉伯糖溶解在1ml MilliQ水中以产生10mg/ml溶液
    向900μlMilliQ水中加入100μl10 mg/ml阿拉伯糖溶液,得到1 mg/ml溶液
    根据下表2制备5种阿拉伯糖溶液

    表2.阿拉伯糖溶液制备
    阿拉伯糖浓度,μg/ml
    体积为1 mg/ml阿拉伯糖溶液,μl
    MilliQ的体积,微博
    2
    2
    998
    3
    3
    997
    5
    5
    995
    8
    8
    992
    10
    10
    990

致谢

该方案改编自以前发表的研究(Kozlova等人,2015)。这项工作部分支持俄罗斯基础研究基金会(项目## 14-04-01002和15-04-02560)。

参考文献

  1. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法。 Anal Biochem 72:248-254。
  2. Fry,S.C。(2004)。 原代细胞壁代谢:跟踪生命中壁聚合物的职业生涯植物细胞。新植物 161:641-675
  3. Kozlova,L.V.,Gorshkov,O.V.,Mokshina,N.E.and Gorshkova,T.A。(2015)。 玉米中α-L-阿拉伯呋喃糖苷酶的差异表达( Zea mays L 。)根伸长。 241(5):1159-1172。
  4. Kozlova,L.V.,Snegireva A.V.和Gorshkova T.A.(2012)。 混合连接葡聚糖在玉米根茎伸长不同阶段的分布和结构 Russ J Plant Phys 59:339-347。
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引用:Kozlova, L. V., Mikshina, P. V. and Gorshkova, T. A. (2016). Assay of Arabinofuranosidase Activity in Maize Roots. Bio-protocol 6(6): e1764. DOI: 10.21769/BioProtoc.1764.
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