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Phenol-based Total Protein Extraction from Lily Plant Tissues
从百合组织中提取苯酚标记总蛋白   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
May 2014

Abstract

The phenol-based total protein extraction method is unique in that water-soluble components such as polyphenolic compounds and nucleic acids can be easily removed. Thus, total protein is free from contaminants and allows for high quality two–dimensional gel electrophoresis. The phenol-based extraction of total protein was used in various lily organs and may likely apply to other plants whose content of polyphenolics is high (Note 1). An additional advantage of this extraction method is that nucleic acids can be easily removed and thus, avoid adverse effects of nucleic acids on protein resolution in the gel. This method is modified from that of Hurkman and Tanaka (1986).

Keywords: Protein extratction (蛋白质extratction), Phenol (苯酚), Plant (植物)

Materials and Reagents

  1. Sucrose (United States Biological, catalog number: 21938 )
  2. Tris base (United States Biological, catalog number: 75825 )
  3. HCl (fuming, at 37%) (Merck KGaA, catalog number: 100317 )
  4. EDTA (United States Biological, catalog number: 15699 )
  5. KCl (SERVA, catalog number: 26868 )
  6. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  7. Phenol (Merck KGaA, catalog number: 100206 )
  8. NH4OAc (Merck KGaA, catalog number: 101116 )
  9. 100% MeOH (J.T. Baker®, catalog number: 9093-03 )
  10. Urea (United States Biological, catalog number: 75826 )
  11. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  12. K2CO3 (Merck KGaA, catalog number: 106683 )
  13. L-lysine (Sigma-Aldrich, catalog number: L5501 )
  14. DTT (Merck KGaA, catalog number: 111474 )
  15. Extraction buffer (see Recipes)
  16. Dissolving buffer (see Recipes)
  17. 0.1 M NH4OAc in 100% MeOH (see Recipes)

Equipment

  1. Ceramic mortar and pestles
  2. Beaker
  3. Stir bar
  4. Centrifuge (Eppendorf, model: 5424 )
  5. AVANTI® J-26 XP High-Performance centrifuge with a JA 25.50 fixed-angle rotor (Beckman Coulter)
  6. COREX® tube,DuPont Instruments (catalog number: 00152 )
  7. 1.5 ml Eppendorf tubes
  8. Fume hood
  9. Speed vacuum (Savant Systems LLC, model: SC110 )
  10. Mixer (Vortex Genie 2, Scientific Industries, Inc.)
  11. Stirrer (Corning)

Procedure

  1. Lily plant tissue collection
    1. Various vegetative and floral organs of young lily plants, including flower buds with a length around 4 cm were collected. The first three whorls of young leaves around the buds, plus the entire roots (approximately 8 cm from the apex) were also collected.
    2. Each organ was dissected from lily plants in the field and immediately frozen in liquid nitrogen and stored at -80 °C.

  2. Homogenization
    1. Work in the fume hood. Add liquid nitrogen into a mortar (12.5 cm) and pestle and keep them frozen.
    2. Add and grind 1 g of fresh or frozen tissue into powder in the frozen mortar and pestle in liquid nitrogen. Keep the sample frozen at all times.
    3. Add 5 ml of water-saturated phenol and 5 ml of extraction buffer to the sample powder.
    4. Add more liquid nitrogen into the mortar for easy grinding and mixing of the plant sample. Repeat one more time.
    5. Leave the frozen sample mix in the mortar until it melts and turns into a slurry.
    6. Grind the sample mix vigorously as soon as it turns into slurry.

      Video 1. Homogenization of lily plant tissues

    7. Transfer the melting solution into a glass beaker (25 or 50 ml in volume) and gently mix with a stir bar for 30 min at room temperature.

  3. Phase separation-partition of protein into the phenol phase
    1. Transfer the sample mixture into a COREX® tube.
    2. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    3. Transfer the upper phase (phenol phase) into a new 50 ml tube.
    4. Add an equal volume of phenol to the aqueous phase.
    5. Mix thoroughly by vigorous vortexing for 1 min.
    6. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    7. Remove and pool the upper phase (phenol phase).

  4. Phase separation-repetitive removal of contaminants from the phenol phase
    1. Add an equal volume of extraction buffer to the pooled phenol phase.
    2. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    3. Transfer the upper phase (phenol phase) into a new 50 ml tube.

  5. Protein precipitation
    1. Add 5x volume of 0.1 M NH4OAc in 100% MeOH (cold) and mix well by vortexing for 1 min.
    2. Precipitate protein at -20 °C for 2 h or overnight (approximately 16 h) (Note 2).
    3. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    4. Decant the supernatant.

  6. Protein wash
    1. The protein pellet is added with 10 ml of 0.1 M NH4OAc in 100% MeOH (cold).
    2. Wash the pellet by vigorous vortexing.
    3. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    4. Repeat the washing step twice.
    5. Decant the supernatant.
    6. Add 10 ml of acetone.
    7. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    8. Repeat the washing step twice.
    9. The pellet is shortly dried by speed vacuum for 30-60 sec.
    10. Dissolve the pellet in 300-500 μl (Note 3) of dissolving buffer and mix constantly by vortexing for 30 min. If the pellet is big, break it down into small pieces with a glass bar in order to increase protein solubility.
    11. Centrifuge at 10,000 rpm for 10 min at 4 °C.
    12. Collect supernatant (protein). The pellet (Note 4) in the tube is redissolved in 300-500 μl of dissolving buffer.
    13. This process of protein dissolution may be repeated several times until the concentration of protein in the dissolving buffer becomes insignificant.
    14. The collected supernatant may be pooled together.

Representative data



Figure 1. Anther-specificity analysis of lily protein by SDS-PAGE. Total protein (40 μg) was isolated from various organs of L. longiflorum, separated by 12% SDS-PAGE and stained with Coomassie blue. 1, root; 2, stem; 3, leaf; 4, tepal; 5, filament; 6, anther; 7, pistil; M, marker.


Figure 2. 2D-PAGE analysis of lily pollen protein. Total protein (1.5 mg) was isolated from pollen of L. longiflorum, separated by 2D-PAGE and stained with Coomassie blue. Marker proteins are indicated in the left.

Recipes

  1. Extraction buffer

    10 ml
    Final concentration
    Sucrose
    2.4 g
    0.7 M
    Tris base
    0.61 g
    0.5 M
    EDTA
    0.19 g
    50 mM
    HCl fuming 37%
    24.8 μl
    30 mM
    1.0 M KCl
    1 ml
    100 mM
    2-Mercaptoethanol
    200 μl
    2%
    Then fill ddH2O up to 10 ml.

  2. Dissolving buffer

    3 ml
    Final concentration
    Urea
    1.7 g
    9.5 M
    10% Triton X-100
    600 μl
    2%
    2.5 M K2CO3
    6 μl
    5 mM
    L-lysine
    1.5 mg
    500 μg/ml
    1 M (15.4%) DTT
    97.4 μl
    0.5 %
    ddH2O is added to a volume of 3 ml, filter-sterilized (0.22 μm), aliquoted and stored at -20 °C.

  3. 0.1 M NH4OAc in 100% MeOH
    0.771 g of NH4OAc (MW = 77.08) is added into 100 ml of 100 % MeOH

Notes

  1. In our experience, total protein extracted from various organs of Arabidopsis and Tobacco plants using the phenol-based total protein extraction method yields high quality protein.
  2. Protein precipitated at -20 °C overnight (approximately 16 h) may allow a better yield of total protein. However, if yield is not a concern, most proteins can be precipitated at -20 °C for 2 h.
  3. It is important to maintain protein samples in a high concentration (at least 15 μg/μl) given that the maximum of sample loading in isolectric focusing of 2D-PAGE is around 100 μl. If protein is prepared for SDS-PAGE only, a larger volume of dissolving buffer can be applied.
  4. Protein in the pellet can be dissolved with the dissolving buffer. However, insoluble materials may remain in the pellet after centrifugation.

Acknowledgments

The protocol is mainly modified from that of Hurkman and Tanaka (1986). We appreciate their original contribution.

References

  1. Hurkman, W. J. and Tanaka, C. K. (1986). Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol 81(3): 802-806.
  2. Wang, C. S., Walling, L. L., Eckard, K. J. and Lord, E. M. (1992). Patterns of protein accumulation in developing anthers of Lilium longiflorum correlate with histological events. Am J Bot. 118-127.
  3. Yang, C. Y., Chen, Y. C., Jauh, G. Y. and Wang, C. S. (2005). A Lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiol 139(2): 836-846.

简介

酚类总蛋白提取方法的独特之处在于可以容易地除去多酚化合物和核酸等水溶性成分。 因此,总蛋白不含污染物,并允许高质量的二维凝胶电泳。 总蛋白的苯酚提取用于各种百合器官,可能适用于多酚含量高的其他植物(注1)。 该提取方法的另一个优点是,可以容易地除去核酸,从而避免核酸对凝胶中蛋白质分辨的不利影响。 这种方法从Hurkman和Tanaka(1986)的修改。

关键字:蛋白质extratction, 苯酚, 植物

材料和试剂

  1. 蔗糖(United States Biological,目录号:21938)
  2. Tris碱(United States Biological,目录号:75825)
  3. HCl(发烟,37%)(Merck KGaA,目录号:100317)
  4. EDTA(United States Biological,目录号:15699)
  5. KCl(SERVA,目录号:26868)
  6. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  7. 苯酚(Merck KGaA,目录号:100206)
  8. NH 4 OAc(Merck KGaA,目录号:101116)
  9. 100%MeOH(J.T.Baker,,目录号:9093-03)
  10. Urea(United States Biological,目录号:75826)
  11. Triton X-100(Sigma-Aldrich,目录号:T9284)

  12. (Merck KGaA,目录号:106683)
  13. L-赖氨酸(Sigma-Aldrich,目录号:L5501)
  14. DTT(Merck KGaA,目录号:111474)
  15. 提取缓冲液(参见配方)
  16. 溶解缓冲液(参见配方)
  17. 0.1M NH 4 OAc在100%MeOH中的溶液(参见配方)

设备

  1. 陶瓷砂浆和杵
  2. 烧杯
  3. 搅拌棒
  4. 离心机(Eppendorf,型号:5424)
  5. AVANTI ® J-26 XP高性能离心机,配备JA 25.50固定角转子(Beckman Coulter)
  6. COREX 管,DuPont Instruments(目录号:00152)
  7. 1.5 ml Eppendorf管
  8. 通风橱
  9. 高速真空(Savant Systems LLC,型号:SC110)
  10. 混合器(Vortex Genie 2,Scientific Industries,Inc。)
  11. 搅拌器(Corning)

程序

  1. 百合植物组织收集
    1. 年轻百合植物的各种植物和花器官,包括                      收集长度约4cm的花芽。 前三个                      轮子的年轻叶子在芽附近,加上整个根                      (距离顶点约8cm)也被收集
    2. 在现场从百合植物中分离每个器官,并立即在液氮中冷冻并储存在-80℃。

  2. 均质化
    1. 在通风橱工作。 将液氮加入研钵(12.5厘米)中,并杵保持冷冻
    2. 添加并研磨1克新鲜或冷冻的组织成粉末                      冷冻的研钵和杵在液氮中。 保持样品冷冻                      一直。
    3. 向样品粉中加入5毫升水饱和的苯酚和5毫升提取缓冲液
    4. 向砂浆中加入更多的液氮,以便于研磨和混合植物样品。 重复一次。
    5. 将冷冻的样品混合物留在研钵中,直到其熔化并变成浆料。
    6. 一旦变成浆料,就要剧烈地研磨样品混合物
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    7. 将熔融溶液转移到玻璃烧杯中(25或50ml)                      体积),并在室温下用搅拌棒轻轻混合30分钟。

  3. 相分离 - 将蛋白质分配到苯酚相中
    1. 将样品混合物转移到COREX 管中
    2. 在4℃下以10,000rpm离心10分钟
    3. 将上层相(苯酚相)转移到新的50ml管中
    4. 向水相中加入等体积的苯酚
    5. 通过剧烈涡旋混合彻底1分钟。
    6. 在4℃下以10,000rpm离心10分钟
    7. 取出并混合上层相(苯酚相)。

  4. 相分离 - 从苯酚相中重复去除污染物
    1. 向混合的苯酚相中加入等体积的提取缓冲液
    2. 在4℃下以10,000rpm离心10分钟
    3. 将上层相(苯酚相)转移到新的50ml管中。

  5. 蛋白沉淀
    1. 在100%MeOH(冷的)中加入5倍体积的0.1M NH 4 OAc,并通过涡旋混合1分钟。
    2. 在-20℃下沉淀蛋白2小时或过夜(约16小时)(注2)
    3. 在4℃下以10,000rpm离心10分钟
    4. 倾析上清液。

  6. 蛋白洗涤
    1. 向蛋白质沉淀中加入在100%MeOH(冷)中的10ml 0.1M NH 4 OAc。
    2. 通过剧烈涡旋洗涤沉淀
    3. 在4℃下以10,000rpm离心10分钟。
    4. 重复洗涤步骤两次。
    5. 滗析上清液。
    6. 加入10ml丙酮。
    7. 在4℃下以10,000rpm离心10分钟
    8. 重复洗涤步骤两次。
    9. 丸粒用速度真空短时间干燥30-60秒。
    10. 将沉淀溶解在300-500μl(注3)溶解缓冲液中                      通过涡旋混合30分钟。 如果丸是大的,打破它                      用玻璃棒下降成小块,以增加蛋白质 溶解度。
    11. 在4℃下以10,000rpm离心10分钟
    12. 收集上清液(蛋白质)。 将管中的沉淀(注4)重新溶解在300-500μl溶解缓冲液中
    13. 这种蛋白质溶解过程可以重复几次                      直到溶解缓冲液中的蛋白质浓度变为                      无效。
    14. 可以将收集的上清液合并在一起。

代表数据



图1.通过SDS-PAGE的百合蛋白的花药特异性分析从L的各种器官中分离总蛋白(40μg)。 通过12%SDS-PAGE分离并用考马斯蓝染色。 1,根; 2,茎; 3,叶; 4,tepal; 5,细丝; 6,花药; 7,雌蕊; M,标记。


图2.百合花粉蛋白的2D-PAGE分析从L的花粉分离总蛋白(1.5mg)。 通过2D-PAGE分离并用考马斯蓝染色。 标记蛋白在左侧显示。

食谱

  1. 提取缓冲区

    10 ml
    最终浓度
    蔗糖
    2.4克
    0.7 M
    Tris碱
    0.61克
    0.5 M
    EDTA
    0.19克
    50 mM
    HCl发烟37%
    24.8微升
    30 mM
    1.0 M KCl
    1 ml
    100 mM
    2-巯基乙醇
    200μl
    2%
    然后将ddH sub 2 O填充至10ml。

  2. 溶解缓冲区

    3 ml
    最终浓度
    尿素
    1.7克
    9.5 M
    10%Triton X-100 600μl
    2%
    2.5 M K sub 2 CO 3
    6微升
    5 mM
    L-赖氨酸 1.5 mg
    500μg/ml
    1 M(15.4%)DTT
    97.4μl
    0.5%
    ddH 2 O加入到3ml体积中,过滤灭菌(0.22μm),分装并储存在-20℃。

  3. 0.1M NH 4 OAc在100%MeOH中的溶液 将0.771g的NH 4 OAc(MW = 77.08)加入到100ml的100%MeOH中

笔记

  1. 根据我们的经验,使用基于苯酚的总蛋白提取方法从拟南芥和烟草植物的各种器官提取的总蛋白产生高质量的蛋白质。
  2. 在-20℃下沉淀过夜(约16小时)的蛋白质可允许更好的总蛋白产量。 然而,如果产量不是问题,大多数蛋白质可以在-20℃下沉淀2小时
  3. 由于2D-PAGE的等电聚焦中样品上样量的最大值约为100μl,因此重要的是保持蛋白质样品在高浓度(至少15μg/μl)。 如果仅为SDS-PAGE制备蛋白质,可以使用更大体积的溶解缓冲液
  4. 可以用溶解缓冲液溶解沉淀中的蛋白质。 然而,离心后不溶物可能留在沉淀中。

致谢

该协议主要修改自Hurkman和Tanaka(1986)。 我们感谢他们的最初贡献。

参考文献

  1. Hurkman,W.J.and Tanaka,C.K。(1986)。 通过二维凝胶电泳分析植物膜蛋白的溶解性。 植物生理学 81(3):802-806
  2. Wang,C.S.,Walling,L.L.,Eckard,K.J.and Lord,E.M。(1992)。 Lilium longiflorum发育的花药中蛋白质积累的模式< em>与组织学事件相关联。 118-127。
  3. Yang,C.Y.,Chen,Y.C.,Jauh,G.Y.and Wang,C.S。(2005)。 Lily ASR蛋白涉及脱落酸信号传导,赋予拟南芥中的抗旱和抗盐性。 植物生理学 139(2):836-846。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y., Hsiao, Y. and Wang, C. (2015). Phenol-based Total Protein Extraction from Lily Plant Tissues. Bio-protocol 5(4): e1399. DOI: 10.21769/BioProtoc.1399.
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