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植物细胞壁物质提取法(微量)

曾文静 |  2022-11-23  | DOI: 10.21769/BioProtoc.v1097
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视频简介该方法使用各种有机溶剂去除色素,蛋白,脂质等物质。是一种提取快速醇不溶性残留物(alcohol-insoluble residue)胞壁物质制备法。
  • 视频介绍

一、视频摘要

本实验方法为快速酒精不溶性残留物制备法,首先用冰酒精使样品中的酶失活和溶解色素、其次多次使用酒精和丙酮完全除色素, 氯仿/甲醇除脂质,之后为了尽快干燥用甲醇代替之前溶剂,最终获得呈蓬松的白色/灰白色粉末状的细胞壁提取物。

This method is rapid alcohol-insoluble residue preparation method. First, use the cooled-alcohol to dissolve the pigment and inactive the enzymes in samples. Second, use alcohol and acetone for many times to completely remove the pigment, and use chloroform/methanol to remove the lignin. Then, use methanol to replace the previous solvent in order to dry as soon as possible. Finally, obtain the puffy white/gray white powder cell wall extract.


二、关键词

丙酮、快速乙醇不溶性残留物制备法、叶片胞壁

Acetone, Rapid alcohol-insoluble residue (AIR) preparation method, Leaf cell wall


三、实验样品信息,试剂、耗材或仪器

1. 样品信息

青花菜叶片

Broccoli leaf

2. 试剂和耗材

试剂:80%乙醇、丙酮、氯仿/甲醇混合液(1:1,v/v)、甲醇

耗材:2ml离心管

Reagent: 80% ethanol, acetone, chloroform/methanol mixture (1:1, v/v), ethanol

Consumables: 2ml centrifuge tube

3. 仪器和软件

移液枪、离心机、通风橱、冰箱、制冰机

Pipette, Centrifuge, Laboratory chemical hood, Low-temperature refrigerator, Ice machine.


四、实验操作

1.尽量避免使用锥底离心管进行实验,因为不利于涡旋。

2.80%乙醇需提前预冷。

3.乙醇浓度可在70-95%范围内进行调整,样品色素含量较高,建议使用较高的乙醇浓度。

4.胞壁提取物最后的干燥方式也可为:37~40℃烘干至恒重。

1.Try to avoid using a cone bottom centrifuge tube for experiments, because it makes subsequent vortex difficult.

2.80% ethanol shall be precooled.

3.The ethanol concentration can be adjusted within the range of 70-95%. The pigment content of the sample is high, so it is recommended to use a higher ethanol concentration.

4. The drying method of the extract also be dried to constant weight at 37~40 ℃


五、注意事项

1. 因实验中存在大量有机溶剂、实验前请检查通风橱是否正常运转。

2. 确认实验手套是否耐受丙酮、氯仿、甲醇。

3. 植物组织注意充分研磨,过粗颗粒不利于有机溶剂去除色素等非胞壁物质。

1. Since there are a lot of organic solvents in the experiment, please check whether the Laboratory chemical hood operates normally before the experiment.

2. Confirm whether the test gloves are resistant to acetone, chloroform and methanol.

3.The plant tissue shall be fully ground, and the coarse particles are unfavorable to the removal of pigment and other non-cell wall substances by organic solvent.


六、结果分析

最终的细胞壁提取物呈蓬松的白色/灰白色粉末状。

The final cell wall extract was fluffy white/grayish white powder.


七、参考文献

  1. Pettolino FA, Walsh C, Fincher GB and Bacic A, Determining the polysaccharide composition of plant cell walls. Nat Protoc 7:1590-1607 (2012).

  2. Deng, X., et al., Flag leaf cell wall functional groups and components play a crucial role in the accumulation and translocation of Cd in rice grain via foliage application of humic acid. Ecotoxicology and Environmental Safety, 2022. 239: p. 113658.

  3. Hu H, Brown PH and Labavitch JM, Species variability in boron requirement is correlated with cell wall pectin. J Exp Bot 47:227-232 (1996).

  4. Luo J, Ni D, Li C, et al. The relationship between fluoride accumulation in tea plant and changes in leaf cell wall structure and composition under different fluoride conditions[J]. Environmental Pollution, 2021, 270: 116283.

  5. Figueroa C R, Opazo M C, Vera P, et al. Effect of postharvest treatment of calcium and auxin on cell wall composition and expression of cell wall-modifying genes in the Chilean strawberry (Fragaria chiloensis) fruit. Food Chemistry [J], 2012. 132: 2014-2022.

  6. 黄伟男, 枇杷果实采后细胞木质素积累与细胞壁果胶动力学机制研究, 2021, 浙江大学.


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