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Rapid Isolation of Total Protein from Arabidopsis Pollen
从拟南芥花粉中快速分离总蛋白   

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Abstract

Arabidopsis pollen is an excellent system for answering important biological questions about the establishment and maintenance of cellular polarity and polar cell growth, because these processes are amenable to the genetic and genomic approaches that are readily available in Arabidopsis. Given that proteins are the direct executors of a wide variety of cellular processes, it is important to rapidly and efficiently isolate total protein for various protein-based analyses, such as Western blotting, co-immunoprecipitation and mass spectrometry, among others. Here we present a protocol for rapid isolation of total protein from Arabidopsis pollen, which is adapted from our recently published paper (Chang and Huang, 2015).

Keywords: Arabidopsis thaliana(拟南芥), Flower(花), Pollen(花粉), Protein isolation(蛋白质分离), Western blot(蛋白质印迹)

Background

Pollen is a critical stage during the life cycle of sexually-reproducing plants. Pollen germination and subsequent tube growth provides the passage for two non-motile sperm cells to effect double fertilization in flowering plants. Pollen is routinely used as a model system to addressing fundamental cell biological questions, such as the establishment and maintenance of cell polarity and polar cell growth, as well as the structure and function of the actin cytoskeleton (Chen et al., 2009; Qu et al., 2015). Generally, pollen derived from lily and tobacco was widely used due to the large size of the grains, which makes them easy to collect and observe under the microscope. In contrast, Arabidopsis pollen is small and it is relatively difficult to collect a large amount of pollen to isolate a sufficient quantity of protein for downstream analyses such as Western blotting and mass spectrometry. Therefore, development of a protocol to rapidly isolate total protein from Arabidopsis pollen will facilitate related analyses.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes (Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 )
  2. Pipette tips (USA Scientific, catalog numbers: 1112-1720 , 1110-1200 )
  3. Arabidopsis plants (ecotype Col-0)
  4. Ponceau S (Sigma-Aldrich, catalog number: P3504 )
  5. Anti-α-tubulin (Beyotime Biotechnology, catalog number: AT819 )
  6. Anti-actin (Abmart, catalog number: M20009 )
  7. Anti-COXII (Agrisera, catalog number: AS04 053A )
  8. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (AMRESCO, catalog number: 0511 )
  9. K-acetate (Sinopharm Chemical Reagents, catalog number: 30154518 )
  10. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma, catalog number: 63064 )
  11. Tween 20 (AMRESCO, catalog number: 0777 )
  12. Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
  13. Phenylmethanesulfonyl fluoride (PMSF) (EMD Millipore, catalog number: 52332 )
  14. K-HEPES protein extraction buffer (see Recipes)

Equipment

  1. Pestles (VWR, catalog number: 89093-446 )
  2. Forceps (Shanghai Haiou)
  3. Pipettes (VWR, catalog numbers: 89079-970 , 89079-974 )
  4. Vortex (Corning, model: Corning® LSETM Vortex Mixer )
  5. Centrifuge (Eppendorf, model: 5417 R )

Procedure

  1. Grow Arabidopsis plants in a growth room at 21 °C under long-day conditions (16 h light/8 h dark) for 6 to 8 weeks. Normally, the Col-0 ecotype will produce many flowers under these conditions. The plant growth time can be changed to adapt to the flowering time of other Arabidopsis ecotypes.
  2. Quickly collect more than 100 freshly opened flowers (stage 13-15, Figure 1) in one 1.5 ml Eppendorf tube and add 1 ml K-HEPES protein extraction buffer (see Recipes). This step can be done at room temperature.
    Note: Collect Arabidopsis flowers around 10 AM. At this time the flowers will be fully opened, allowing easier access to the pollen.


    Figure 1. An opening Arabidopsis flower at stage 13-15 (Müller, 1961; Smyth et al., 1990)

  3. Vortex the flowers at the highest speed for 2-5 min in order to thoroughly separate the pollen from the flowers (Video 1).

    Video 1. Procedure for the isolation of pollen from flowers by vortex in step 3

    To play the video, you need to install a newer version of Adobe Flash Player.

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  4. Centrifuge at 350 x g for 1 min at 4 °C (Video 2).
    Note: Under low speed, most of the flowers will be suspended in the upper layer of buffer, but the pollen will be centrifuged to the bottom of the tube.

    Video 2. Procedure for the isolation of protein as stated in step 4-7

    To play the video, you need to install a newer version of Adobe Flash Player.

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  5. Slowly and thoroughly remove all the flower debris with small forceps (Video 2).
    Note: If necessary, you can repeat steps 4 and 5 several times for total removal of the flower debris, but you should be careful to avoid touching the pollen at the bottom of the tube.
  6. Completely remove the supernatant (Video 2).
    Note: Make sure the pollen is still at the bottom of the tube.
  7. Put the tube on ice and thoroughly grind the pollen with a small pestle for 1-3 min (Video 2). The liquid mixture will turn yellow color if the pollen is homogenized completely.
    Note: If your lab doesn’t have suitable pestles for 1.5 ml tubes, you can use a 1 ml pipette tip, sealed by melting it in a flame, as a substitute.
  8. Add 50 μl K-HEPES protein extraction buffer into the tube and keep the tube on ice for about 1 h.
  9. Centrifuge at 18,000 x g for 20 min at 4 °C, then transfer the supernatant to a new 1.5 ml tube (keep the tube on ice). Repeat this step twice. The supernatant is the final pollen protein sample and the protein concentration can be measured by standard Bradford assay.

Data analysis

SDS-PAGE and Western blot analyses of Arabidopsis pollen total proteins are shown in Figure 2. Total proteins isolated from Arabidopsis rosette leaves, in which the small subunit of Rubisco is by far the most abundant component, were used as the control (Figure 2A). In contrast, the band corresponding to the small subunit of Rubisco is hardly detected in the pollen total proteins (Figure 2B). This means that detection of proteins other than the small subunit of Rubisco might be easier in Arabidopsis total protein extract. For instance, by loading 10 μg of pollen total proteins per lane, α-tubulin (TUA), actin (ACT), cytochrome c oxidase subunit II (COXII) and profilin (PRF) can be easily detected (Figure 2B).


Figure 2. SDS-PAGE analysis of total proteins isolated from Arabidopsis pollen and leaves, and Western blot analysis of Arabidopsis pollen total proteins probed with several antibodies. A. SDS-PAGE analysis of total proteins (50 μg) isolated from Arabidopsis rosette leaves. The nitrocellulose (NC) membrane was stained with Ponceau S after protein transfer. B. The left panel shows the SDS-PAGE analysis of total proteins (10 μg) isolated from Arabidopsis pollen. The NC membrane was stained with Ponceau S after protein transfer. The right panel shows the Western blot analysis of Arabidopsis pollen total proteins probed with anti-α-tubulin, anti-actin, anti-COXII and profilin antibodies. The profilin antibody was generated in our laboratory; see the details in our published paper (Liu et al., 2015). Other antibodies are commercially available: anti-α-tubulin (Beyotime Biotechnology, AT819), anti-actin (Abmart, M20009) and anti-COXII (Agrisera, AS04 053A).

Notes

Generally, 100 Col-0 flowers will yield about 100 μg total pollen protein with this method.

Recipes

  1. K-HEPES protein extraction buffer
    20 mM HEPES, pH 7.0
    110 mM K-acetate
    2 mM MgCl2
    0.1% Tween 20
    0.2% Triton X-100
    1 mM PMSF (add immediately before using)
    Note: This buffer (without the PMSF) can be stored for up to 6 months at 4 °C.

Acknowledgments

This protocol was modified from our previously published work (Chang and Huang, 2015). The work in Huang’s lab was supported by funding from the Ministry of Science and Technology of China (2013CB945100) and the National Natural Science Foundation of China (31671390 and 31471266).

References

  1. Chang, M. and Huang, S. (2015). Arabidopsis ACT11 modifies actin turnover to promote pollen germination and maintain the normal rate of tube growth. Plant J 83(3): 515-527.
  2. Chen, N., Qu, X., Wu, Y. and Huang, S. (2009). Regulation of actin dynamics in pollen tubes: control of actin polymer level. J Integr Plant Biol 51(8): 740-750.
  3. Liu, X., Qu, X., Jiang, Y., Chang, M., Zhang, R., Wu, Y., Fu, Y. and Huang, S. (2015). Profilin regulates apical actin polymerization to control polarized pollen tube growth. Mol Plant 8(12): 1694-1709.
  4. Müller, A. (1961). Zur Charakterisierung der Blüten und Infloreszenzen von Arabidopsis thaliana (L.) Heynh. Kulturpflanze 9(1): 364-393.
  5. Qu, X., Jiang, Y., Chang, M., Liu, X., Zhang, R. and Huang, S. (2015). Organization and regulation of the actin cytoskeleton in the pollen tube. Front Plant Sci 5: 786.
  6. Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M. (1990). Early flower development in Arabidopsis. Plant Cell 2(8): 755-767.

简介

拟南芥花粉是用于回答关于细胞极性和极性细胞生长的建立和维持的重要生物学问题的优秀系统,因为这些方法适用于在中容易获得的遗传和基因组方法, 拟南芥。 鉴于蛋白质是各种细胞过程的直接执行者,重要的是快速有效地分离各种基于蛋白质的分析的总蛋白质,例如Western印迹,共免疫沉淀和质谱等。 在这里,我们提出了一种从拟南芥花粉中快速分离总蛋白的方案,该方法是从我们最近发表的论文(Chang and Huang,2015)中得到改编的。
花粉是性繁殖植物生命周期的关键阶段。花粉萌发和随后的管生长提供了两个非活动精子细胞在开花植物中进行双重受精的通道。花粉通常被用作解决基本细胞生物学问题的示范系统,例如细胞极性和极细胞生长的建立和维持,以及肌动蛋白细胞骨架的结构和功能(Chen等人, ,2009; Qu等人,2015)。通常来自百合和烟草的花粉由于粒度大而被广泛使用,这使得它们在显微镜下容易收集和观察。相比之下,拟南芥花粉小,收集大量花粉以分离足够量的蛋白质以进行下游分析(如Western印迹和质谱法)相对困难。因此,开发快速分离总拟蛋白质拟南芥花粉的方案将有助于相关分析。

关键字:拟南芥, 花, 花粉, 蛋白质分离, 蛋白质印迹

材料和试剂

  1. 1.5ml Eppendorf管(Fisher Scientific,Fisherbrand TM,目录号:05-408-129)
  2. 移液器提示(USA Scientific,目录号:1112-1720,1110-1200)
  3. 拟南芥植物(生态型Col-0)
  4. Ponceau S(Sigma-Aldrich,目录号:P3504)
  5. 抗α-微管蛋白(Beyotime Biotechnology,目录号:AT819)
  6. 抗肌动蛋白(Abmart,目录号:M20009)
  7. 抗COXII(Agrisera,目录号:AS04 053A)
  8. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(AMRESCO,目录号:0511)
  9. 醋酸钾(国药化学试剂,目录号:30154518)
  10. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma,目录号:63064)
  11. 吐温20(AMRESCO,目录号:0777)
  12. Triton X-100(Sigma-Aldrich,目录号:V900502)
  13. 苯基甲磺酰氟(PMSF)(EMD Millipore,目录号:52332)
  14. K-HEPES蛋白提取缓冲液(参见食谱)

设备

  1. 杵(VWR,目录号:89093-446)
  2. 镊子(上海海鸥)
  3. 移液器(VWR,目录号:89079-970,89079-974)
  4. 涡旋(Corning,型号:Corning ® LSE TM 涡旋混合器)
  5. 离心机(Eppendorf,型号:5417 R)

程序

  1. 在长达21天的条件下(16小时光/8小时黑暗)在21℃的生长室中种植拟南芥植物6至8周。通常,Col-0生态型将在这些条件下产生许多花。可以改变植物生长时间以适应其他拟南芥生态型的开花时间。
  2. 在一个1.5ml Eppendorf管中快速收集100多个新鲜开花(图13-15,图1),并加入1ml K-HEPES蛋白提取缓冲液(参见食谱)。这个步骤可以在室温下完成。
    注意:上午10点左右收集拟南芥花。此时,花朵将被完全打开,可以更容易地进入花粉。


    图1.第13-15阶段的开花拟南芥花/(Müller,1961; Smyth等,1990)

  3. 以最高速度旋转花朵2-5分钟,以便将花粉与鲜花完全分开(视频1)。

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    视频1.在步骤3中通过涡流从花中分离花粉的步骤
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  4. 在4℃下以350 x g离心1分钟(视频2)。
    注意:在低速下,大多数花朵将悬浮在上层缓冲液中,但花粉将被离心至管底。

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    视频2.如步骤4-7所述分离蛋白质的步骤
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  5. 用小镊子缓慢地彻底清除所有的花卉碎片(视频2)。
    注意:如有必要,您可以重复步骤4和5,以完全去除花粉碎物,但您应注意避免接触管底部的花粉。
  6. 彻底清除上清液(视频2) 注意:确保花粉仍在管的底部。
  7. 将管放在冰上,用小杵彻底研磨花粉1-3分钟(视频2)。如果花粉完全匀浆,液体混合物会变黄 注意:如果您的实验室没有1.5毫升管的合适的杵,您可以使用1毫升吸头,将其熔化在火焰中,作为替代物。
  8. 将50μlK-HEPES蛋白提取缓冲液加入管中,并将管保持在冰上约1 h
  9. 在4℃下以18,000xg离心20分钟,然后将上清液转移到新的1.5ml管中(将管保持在冰上)。重复此步骤两次。上清液是最终的花粉蛋白质样品,蛋白质浓度可以通过标准Bradford测定法测定

数据分析

拟南芥花粉总蛋白的SDS-PAGE和Western印迹分析显示在图2中。从拟南芥分离的总蛋白质玫瑰花叶,其中Rubisco的小亚基是远最丰富的组分被用作对照(图2A)。相比之下,在花粉总蛋白中几乎不检测到与Rubisco的小亚基相对应的条带(图2B)。这意味着在拟南芥总蛋白提取物中检测Rubisco小亚基以外的蛋白质可能更容易。例如,通过每泳道加载10μg花粉总蛋白,可以容易地检测到α-微管蛋白(TUA),肌动蛋白(ACT),细胞色素c氧化酶亚基II(COXII)和profilin(PRF)(图2B)。 />

图2.从拟南芥花粉和叶分离的总蛋白质的SDS-PAGE分析,以及用几种抗体探测的拟南芥花粉总蛋白的蛋白质印迹分析强> A.从拟南芥分离的总蛋白(50μg)的玫瑰花叶的SDS-PAGE分析。蛋白转移后,硝基纤维素(NC)膜用Ponceau S染色。 B.左图显示从拟南芥花粉分离的总蛋白(10μg)的SDS-PAGE分析。蛋白转移后,NC膜用Ponceau S染色。右图显示用抗α-微管蛋白,抗肌动蛋白,抗COXII和profilin抗体探测的拟南芥花粉总蛋白的蛋白质印迹分析。 profilin抗体在我们的实验室中产生;请参阅我们发表的论文(Liu等人,2015年)中的细节。其他抗体可商购:抗α-微管蛋白(Beyotime Biotechnology,AT819),抗肌动蛋白(Abmart,M20009)和抗COXII(Agrisera,AS04 053A)。

笔记

一般来说,100个Col-0花将通过该方法产生约100μg总花粉蛋白。

食谱

  1. K-HEPES蛋白提取缓冲液
    20mM HEPES,pH 7.0
    110 mM醋酸钾 2mM MgCl 2
    0.1%吐温20
    0.2%Triton X-100
    1毫升PMSF(使用前立即添加)
    注意:此缓冲液(不含PMSF)可在4°C下储存长达6个月。

致谢

这个协议是从我们以前发表的作品中修改的(Chang and Huang,2015)。黄实验室的工作得到了中国科技部(2013CB945100)和国家自然科学基金(31671390和31471266)的资助。

参考文献

  1. Chang,M.和Huang,S。(2015)。拟南芥 ACT11修饰肌动蛋白周转率以促进花粉萌发并维持正常的管生长速率。植物J 83(3):515-527。 />
  2. Chen,N.,Qu,X.,Wu,Y.and Huang,S。(2009)。花粉管中肌动蛋白动力学的调节:肌动蛋白聚合物水平的控制.J Integr Plant Biol 51(8):740-750。 br />
  3. Liu,X.,Qu,X.,Jiang,Y.,Chang,M.,Zhang,R.,Wu,Y.,Fu,Y.and Huang,S。(2015)。< a class = profilin调节顶端肌动蛋白聚合以控制极性花粉管生长。 莫尔工厂 8(12):1694-1709。
  4. Müller,A.(1961)。  Zur Charakterisierung derBlütenund Infloreszenzen von 拟南芥(L.)Heynh。 Kulturpflanze 9(1):364-393。
  5. Qu,X.,Jiang,Y.,Chang,M.,Liu,X.,Zhang,R.and Huang,S。(2015)。  花粉管中肌动蛋白细胞骨架的组织和调控。前植物科学 5:786 。
  6. Smyth,DR,Bowman,JL和Meyerowitz,EM(1990)。拟南芥中的早期花卉开发 植物细胞 2(8):755-767。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chang, M. and Huang, S. (2017). Rapid Isolation of Total Protein from Arabidopsis Pollen. Bio-protocol 7(8): e2227. DOI: 10.21769/BioProtoc.2227.
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