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Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
拟南芥幼苗中细胞溶质,微粒体,游离多核糖体(FP)和膜结合多聚体(MBPs)的分离   

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Abstract

The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate the cytosol, microsome, free polysome (FP) and membrane bound polysome (MBP) from plant tissue. The isolated fractions are suitable for genome wide profiling of mRNAs, small RNAs and proteins.

Keywords: Cytosol(胞液), Microsome(微粒体), Free polysome(游离多核糖体), Microsome bound polysome(微粒体结合的多核糖体)

Background

Plant endomembrane system is very important for cell wall formation, lipid biosynthesis, protein synthesis, modification, folding and trafficking. According to the co-translational translocation model, signal peptides at the N-terminal of secreted proteins are synthesized by cytosolic polysomes, and then recognized by signal recognition particles on ER, and the remaining portion of proteins will be subsequently synthesized on ER. According to this model, only mRNAs encoding for secreted proteins could be brought to ER for translation (Peter and Johnson, 1994). However, large portion of mRNAs were identified from mammalian and plant cell ERs (Lerner et al., 2003; de Jong et al., 2006), and recent studies revealed that ER also functions as a key hub for small RNA function in plant (Li et al., 2013 and 2016). These findings broadened our knowledge about ER functionality. Large scale identification of mRNAs, small RNAs and proteins from ER of cells upon different developmental stages and environmental stimuli will provide valuable clues for elucidating new functions of ER. Here, we describe a protocol to isolate the cytosol, microsome, FP and MBP from Arabidopsis thaliana, and it could be adapted to rice, maize and other plants.

Materials and Reagents

  1. Pipette tip (Denville Scientific, catalog numbers: P2101 , P2102 , P2109 ), autoclave before use
  2. 50 ml tube
  3. Miracloth (EMD Millipore, catalog number: 475855-1R )
  4. 15 ml tube
  5. 13 x 51 mm centrifuge tubes (Beckman Coulter, catalog number: 326819 )
  6. 25 x 89 mm centrifuge tubes (Beckman Coulter, catalog number: 355631 )
  7. Arabidopsis ecotype Columbia-0 maintained by our own laboratory
  8. Murashige and Skoog medium
  9. Liquid nitrogen
  10. 1% (v/v) Triton X-100
  11. DEPC H2O
  12. Tris base (Fisher Scientific, catalog number: BP152-5 )
  13. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A142-212 )
  14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  15. MgOAc
  16. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  17. Sucrose (Fisher Scientific, catalog number: BP220-212 )
  18. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  19. Cycloheximide (Sigma-Aldrich, catalog number: C1988 )
  20. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  21. Ethanol
  22. SUPERaseIN (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2696 )
  23. Magnesium acetate tetrahydrate (MgCl2·4H2O) (Sigma-Aldrich, catalog number: M5661 )
  24. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: BP214-500 )
  25. Proteinase inhibitor cocktail-EDTA free (Roche Diagnostics, catalog number: 18970600 )
  26. Ribosome extraction buffer (see Recipes)
  27. Sucrose cushion buffer (see Recipes)
  28. Resuspending buffers (see Recipes)
  29. 10x sucrose salt (for 15-60% sucrose gradient column) (see Recipes and Notes)

Equipment

  1. Pipette (Eppendorf)
  2. Plant growth chamber (Percival Scientific, model: CU-36L4 )
  3. L8-70M Ultracentrifuge (Beckman Coulter, model: L8-70M )
  4. SW 28 rotor (Beckman Coulter, model: SW 28 Ti )
  5. SW 55 Ti rotor (Beckman Coulter, model: SW 55 Ti )
  6. Type70 Ti rotor (Beckman Coulter, model: Type70 Ti )
  7. 25 x 89 mm bottle, with cap assembly (Beckman Coulter, catalog number: 355618 )
  8. Vacuum pump
  9. Centrifuge (Eppendorf, model: 5424 R )
  10. High speed centrifuge (Beckman Coulter, model: Avanti J-E Series )
  11. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  12. Density gradient fractionation systems (BRANDEL, model: BR-188 )
  13. 37 °C incubator

Software

  1. Data acquisition software (Brandel, model: PEAK CHART)

Procedure

This protocol allows the simultaneous isolation of FP and MBP from the same plant sample. Briefly, the cytosol and microsome fractions are separated by centrifugation, and microsome fraction is dissolved with extraction buffer supplemented with detergent. Both cytosol and microsome lysates are passed through sucrose cushion solution by ultracentrifugation to obtain FP and MBP pellets which are subsequently subjected to density gradient fractionation and profile analyses (Figure 1).


Figure 1. Scheme of FP and MBP isolation

  1. Col-0 seeds are sterilized and plated on Murashige and Skoog medium, and plants are grown in a growth chamber at 23 °C under 16 h light/8 h dark cycles for 12 days.
  2. 2 g seedlings are ground into fine powder in liquid nitrogen, and are suspended in 8 ml ribosome extraction buffer (see Recipes) in a 50 ml tube. Keep on ice for 20 min.
  3.  The slurry is filtered with 2 layers of Miracloth to a 15 ml tube and centrifuged twice at 10,000 x g for 10 min to remove the debris.
  4. The supernatant is transferred into a Beckman centrifuge tube and centrifuged at 30,000 x g for 30 min with a Beckman SW28 rotor. Transfer the supernatant to a new tube as the cytosol fraction and keep it on ice.
  5. Resuspend the pellet with 8 ml ribosome extraction buffer followed by centrifugation at 30,000 x g for another 30 min. Discard the supernatant, and the pellet is kept as the microsome fraction.
    Note: The cytosol and microsome fractions from steps 3 and 4 are ready for RNA and protein extraction. If you want to perform FP/MBP isolation, please continue the following steps.
  6. Dissolve the microsome pellet with 8 ml ribosome extraction buffer supplemented with 1% (v/v) Triton X-100. Keep it on ice for 20 min.
  7. Subject the cytosol extract (step 3) and microsome lysate (step 5) to centrifugation at 30,000 x g for 30 min with a Beckman SW28 rotor to remove any residual membranes.
  8. Transfer 8 ml sucrose cushion solution (see Recipes) into a centrifugation bottle (Beckman centrifuge) suitable for Type70 Ti rotor (Beckman Coulter), and then slowly load the clarified cytosol or microsome lysate from step 6 on the top of the sucrose cushion.
    Note: Be careful not to disturb the sucrose cushion layer.
  9. Centrifuge at 183,960 x g with Type70 Ti rotor (Beckman Coulter) at 4 °C for 3 h.
  10. Draw a circle around the ribosome pellet with a marker pen, and remove all liquid in the tube with a pipette or a vacuum pump (Video 1). Hold the tube with the marked position upward, and carefully wash the inner surface of the tube except for the marked area by 1 ml ddH2O three times with a pipette (Video 1). The purpose of this step is to remove the residual salt and sucrose in the tube. Any touching with the FP/MBP pellet either by pipette tip or water must be avoided.

    Video 1. Removal of supernatant by vacuum and washing of the tube inner wall

  11. Resuspend the pellets in 400 μl resuspension buffer (see Recipes), and transfer them to nuclease free microcentrifuge tubes. Keep the tubes on ice for 30 min.
  12. Centrifuge at 16,000 x g for 5 min at 4 °C to remove debris, and transfer the supernatants to new tubes.
    Note: The samples obtained from step 11 are ready for RNA and protein extraction of FP/MBP. If you want to check the FP/MBP profiles, continue the following steps.
  13. Measure the OD260 of the samples from step 11 with NanoDrop spectrophotometer.
  14. Slowly load 1,000 OD260 of FP or 200 OD260 of MBP on the top of 15-60% sucrose gradient column (see Notes). The yield of MBP is much lower than FP, but 200 OD260 is enough for the MBP profile analysis.
    Note: Be careful not to disturb the sucrose gradient. It is important to keep the pipette tip and the surface of the gradient solution nicely touched (but not protruding into the solution) during the loading, otherwise droplets may be formed and the gradient will be disturbed.
  15. Centrifuge at 237,020 x g with SW55 Ti rotor (Beckman Coulter) for 1.5 h at 4 °C.
  16. Perform the density gradient fractionation. The fractionation system is composed of a syringe pump, a tube piercer stand, a detector, a fraction collector and the Peakchart software (Figure 2A). The gradient column is mounted onto the tube piercer stand and is pierced by the needle at the bottom of stand (see Video 2). For the syringe pump, put the speed mode switch to ‘normal’ position, and the fluid direction switch to ‘off’ position; Turn the control mode knob to ‘remote start/stop’, and adjust the fluid speed to 1.5 ml/min (Figure 2B); For the UA-6 detector, set the sensitivity value as ‘1’, and the chart speed as ‘150 cm/h’ (Figure 2C). The system was under control of the Peakchart software, and ribosome profiles were recorded by the software and the detector (Figure 2D).


    Figure 2. The density gradient fractionation system. A. The overview of the fractionation system. The gradient column is attached to the tube piercer stand, and pierced by the needle at the bottom of the stand. The gradient solution is slowly pushed out from the top of the column by the chase fluid in the syringe pump, and A254 nm absorbance was recorded by the UA-6 detector and the Peakchart software. B. The front panel of the syringe pump. The positions of the switches and knobs reflect the parameter settings during fractionation. C. The front panel of the UA-6 detector. The positions of the knobs reflect the parameter settings during fractionation. D. A screenshot of the Peakchart software. The start or stop of the entire system is controlled by the green button at the bottom center, the profile is shown on the screen in real time manner, and the data are automatically saved when the procedure completes.

    Video 2. Attachment of the gradient column to the density gradient fractionation system

  17. Analyze the ribosome profile. The typical FP/MBP profiles are shown in Figure 3. The different peaks represent 40S small subunit, 60S large subunit, the 80S monoribosome and polyribosomes respectively, and a good isolation of FP or MBP should display a profile with these distinct peaks and a hill shaped pattern instead of a decline curve in the polysome region. Note that the peak of 80S monomer of MBP is much lower than that of FP, and 60S and 80S fractions were usually combined in MBP.


    Figure 3. Profiles of FP and MBP. FP (A) and MBP (B) are separated in 15-60% sucrose gradient by ultracentrifugation, and are fractionated by gradient fractionation system subsequently. The x-axis indicates the sucrose concentration in the corresponding gradient, and the y-axis represents the absorbance level at 254 nm. 40S: small subunit of ribosome; 60S: large subunit of ribosome; 80S: the monoribosome complex.

Notes

Preparation of 15-60% sucrose gradient column. All stock solutions are prepared with DEPC H2O except for CHX and CHL which were prepared with ethanol.

  1. Prepare sucrose solutions with different sucrose concentration (for 10 gradient columns, Table 1):

    Table 1. The recipes for preparing the 15-60% sucrose gradient column


  2. Place 13 x 51 mm centrifuge tubes (Beckman) into a rack that can withstand -80 °C
  3. Start with the 60% sucrose layer, pipette 0.75 ml 60% sucrose solution into a 13 x 51 mm centrifuge tube (Beckman), avoiding any air bubbles, and then freeze for 1h at -80 °C.
  4. Add the next gradient layer with the volumes indicated in the table, freeze again, and continue with the last two layers.
  5. Store the sucrose gradient columns at -80 °C. The gradient columns could be used in 3 months if they are stored properly.
  6. Before use, remove the column from the freezer, and thaw in a 37 °C incubator for exactly 1 h followed by cooling down in a cold room or refrigerator for another 1 h.

Recipes

  1. Ribosome extraction buffer
    0.2 M Tris-HCl, pH 8.5
    0.1 M KCI
    70 mM MgOAc
    50 mM EGTA
    0.25 M sucrose
    10 mM DTT
    50 μg/ml Cycloheximide (CHX) (stock 50 μg/μl in ethanol)
    50 μg/ml Chloramphenicol (CHL) (stock 50 μg/μl in ethanol)
    2.5 U/ml SUPERaseIN
  2. Sucrose cushion solution
    0.4 M Tris-HCl, pH 9.0
    0.2 M KCI
    0.005 M EGTA
    0.035 M MgCl2
    1.75 M sucrose
    5 mM DTT
    50 μg/ml CHX (stock 50 μg/μl in ethanol)
    50 μg/ml CHL (stock 50 μg/μl in ethanol)
  3. Resuspension buffer
    0.2 M Tris-HCl, pH 9.0
    0.2 M KCl
    0.025 M EGTA
    0.035 M MgCl2
    5 mM DTT
    50 μg/ml CHX (stock 50 μg/μl in ethanol)
    50 μg/ml CHL (stock 50 μg/μl in ethanol)
  4. 10x sucrose salt (for 15-60% sucrose gradient column)
    0.4 M Tris-HCl, pH 8.4
    0.2 M KCI
    0.1 M MgCI2

Note: All buffers were prepared with DEPC water if not emphasized; DTT, CHX, CHL and SUPERaseIN need be added freshly.

Acknowledgments

This protocol was adapted from our previous work (Li et al., 2016). We thank Dr. Xuemei Chen for suggestions on this protocol. The work was supported by grants from the science technology and innovation committee of Shenzhen municipality (JCYJ20151116155209176, KQCX2015033110464302, KY20150114), and the Key Laboratory of Shenzhen (ZDSYS20141118170111640).

References

  1. de Jong, M., van Breukelen, B., Wittink, F. R., Menke, F. L., Weisbeek, P. J. and Van den Ackerveken, G. (2006). Membrane-associated transcripts in Arabidopsis; their isolation and characterization by DNA microarray analysis and bioinformatics. Plant J 46(4): 708-721.
  2. Lerner, R. S., Seiser, R. M., Zheng, T., Lager, P. J., Reedy, M. C., Keene, J. D. and Nicchitta, C. V. (2003). Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes. RNA 9(9): 1123-1137.
  3. Li, S., Le, B., Ma, X., Li, S., You, C., Yu, Y., Zhang, B., Liu, L., Gao, L., Shi, T., Zhao, Y., Mo, B., Cao, X. and Chen, X. (2016). Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5.
  4. Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., Ji, L., Pan, Z., Cao, X., Mo, B., Zhang, F., Raikhel, N., Jiang, L. and Chen, X. (2013). MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153(3): 562-574.
  5. Peter, W. and Johnson, A. E. (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 10: 87-119.

简介

植物内膜系统对蛋白质和脂质的合成,修饰和分泌起着至关重要的作用。 从经典观点来看,只有编码分泌蛋白质的mRNA才能通过协同翻译方式靶向内质网(ER)进行翻译,然而最近,这一模型已经被大量的细胞溶质mRNA也可能与 ER,并且一些类别的小RNA在ER上富集。 这些结果表明ER的功能超出了目前的知识。 在微粒体上大规模鉴定RNA和蛋白质对于显示ER功能至关重要,研究将由下一代测序技术提升。 该协议提供了从植物组织中分离细胞质,微粒体,游离多聚体(FP)和膜结合多聚体(MBP)的技术工作流程。 分离的级分适用于mRNA,小RNA和蛋白质的基因组广谱分析。
【背景】植物内膜系统对于细胞壁形成,脂质生物合成,蛋白质合成,修饰,折叠和贩运非常重要。根据共翻译易位模型,分泌蛋白N末端的信号肽由细胞溶质多核糖体合成,然后由ER上的信号识别粒子识别,其余蛋白质部分随后在ER上合成。根据该模型,只有编码分泌蛋白的mRNA可以被带到ER进行翻译(Peter和Johnson,1994)。然而,从哺乳动物和植物细胞ER(Lerner等人,2003; de Jong等人,2006)鉴定了大部分mRNAs,最近的研究显示ER也作为植物中小RNA功能的关键枢纽(Li等人,2013和2016)。这些发现扩大了我们对ER功能的了解。在不同发育阶段和环境刺激下大规模鉴定细胞内的mRNA,小RNA和蛋白质将为阐明ER的新功能提供有价值的线索。在这里,我们描述了从拟南芥分离胞质溶胶,微粒体,FP和MBP的方案,并且可以适应于水稻,玉米和其他植物。

关键字:胞液, 微粒体, 游离多核糖体, 微粒体结合的多核糖体

材料和试剂

  1. 移液器吸头(Denville Scientific,目录号:P2101,P2102,P2109),使用前高压灭菌
  2. 50ml管
  3. Miracloth(EMD Millipore,目录号:475855-1R)
  4. 15毫升管子
  5. 13×51毫米离心管(Beckman Coulter,目录号:326819)
  6. 25 x 89 mm离心管(Beckman Coulter,目录号:355631)
  7. 生物型Columbia-0由我们自己的实验室维护的拟南芥
  8. Murashige和Skoog媒体
  9. 液氮
  10. 1%(v / v)Triton X-100
  11. DEPC H 2 O O
  12. Tris碱(Fisher Scientific,目录号:BP152-5)
  13. 盐酸(HCl)(Fisher Scientific,目录号:A142-212)
  14. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  15. MgOAc
  16. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  17. 蔗糖(Fisher Scientific,目录号:BP220-212)
  18. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  19. 环己酰亚胺(Sigma-Aldrich,目录号:C1988)
  20. 氯霉素(Sigma-Aldrich,目录号:C0378)
  21. 乙醇
  22. SUPERaseIN(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2696)
  23. 乙酸镁四水合物(MgCl 2·4H 2 O)(Sigma-Aldrich,目录号:M5661)
  24. 氯化镁六水合物(MgCl 2·6H 2 O)(Fisher Scientific,目录号:BP214-500)
  25. 蛋白酶抑制剂混合物-EDTA(Roche Diagnostics,目录号:18970600)
  26. 核糖体提取缓冲液(参见食谱)
  27. 蔗糖缓冲垫(见食谱)
  28. 重新安排缓冲区(见配方)
  29. 10x蔗糖盐(15-60%蔗糖梯度柱)(见配方和注释)

设备

  1. 移液器(Eppendorf)
  2. 植物生长室(Percival Scientific,型号:CU-36L4)
  3. L8-70M超速离心机(Beckman Coulter,型号:L8-70M)
  4. SW 28转子(Beckman Coulter,型号:SW 28 Ti)
  5. SW 55 Ti转子(Beckman Coulter,型号:SW 55 Ti)
  6. Type70 Ti转子(Beckman Coulter,型号:Type70 Ti)
  7. 25 x 89毫米瓶,带盖组件(Beckman Coulter,目录号:355618)
  8. 真空泵
  9. 离心机(Eppendorf,型号:5424 R)
  10. 高速离心机(Beckman Coulter,型号:Avanti J-E系列)
  11. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  12. 密度梯度分级系统(BRANDEL,型号:BR-188)
  13. 37℃培养箱

软件

  1. 数据采集软件(Brandel,型号: PEAK CHART

程序

该协议允许从同一植物样品同时隔离FP和MBP。简言之,通过离心分离细胞质和微粒体级分,并用补充有洗涤剂的提取缓冲液溶解微粒体部分。通过超速离心将细胞溶质和微粒体裂解物均通过蔗糖缓冲溶液,得到FP和MBP颗粒,随后进行密度梯度分级和分析(图1)。


图1. FP和MBP隔离的示例

  1. 将Col-0种子灭菌并铺在Murashige和Skoog培养基上,并将植物在23℃,16小时光/ 8小时黑暗循环的生长室中生长12天。
  2. 将2g幼苗在液氮中研磨成细粉末,并在50ml管中悬浮于8ml核糖体提取缓冲液(参见食谱)中。保持在冰上20分钟。
  3. 将浆液用两层miracloth过滤到15ml管中,并以10,000xg离心10分钟以除去碎屑。
  4. 将上清液转移到Beckman离心管中,并用Beckman SW28转子以30,000×g离心30分钟。将上清液转移到新管上,作为细胞质级分,并保持在冰上。
  5. 用8ml核糖体提取缓冲液重新悬浮沉淀,然后以30,000×g离心另外30分钟。弃去上清液,保留颗粒作为微粒体部分 注意:来自步骤3和4的细胞质和微粒体级分已准备好进行RNA和蛋白质提取。如果要执行FP / MBP隔离,请继续执行以下步骤。
  6. 用补充有1%(v / v)Triton X-100的8ml核糖体提取缓冲液溶解微粒体沉淀。保持在冰上20分钟。
  7. 将细胞溶质提取物(步骤3)和微粒体裂解物(步骤5)用Beckman SW28转子以30,000×g离心30分钟以除去任何残留的膜。
  8. 将8毫升蔗糖缓冲溶液(参见食谱)转移到适合Type70 Ti转子(Beckman Coulter)的离心瓶(Beckman离心机)中,然后从蔗糖垫顶部缓慢加载步骤6的澄清的胞质溶胶或微粒体裂解物。
    注意:小心不要打扰蔗糖垫层。
  9. 使用Type70 Ti转子(Beckman Coulter)在4℃下离心至183,960 x g,持续3小时。
  10. 用标记笔在核糖体颗粒周围绘制一圈,并用移液管或真空泵去除管中的所有液体(视频1)。握住带有标记位置的管,并用移液管(视频1)小心地清洗1毫升ddH 2 O 3的标记区域外的管的内表面。该步骤的目的是去除管中残留的盐和蔗糖。必须避免用吸头或水冲击FP / MBP颗粒。

    Video 1. Removal of supernatant by vacuum and washing of the tube inner wall

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  11. 用400μl重悬浮缓冲液(参见食谱)重悬细胞,并将其转移到无核酸酶的微量离心管中。将管保持在冰中30分钟。
  12. 在4℃下以16,000xg离心5分钟以除去碎屑,并将上清液转移到新管中。
    注意:从步骤11获得的样品准备好用于FP / MBP的RNA和蛋白质提取。如果要检查FP / MBP配置文件,请继续执行以下步骤。
  13. 用NanoDrop分光光度计测量来自步骤11的样品的OD 260。
  14. 在15-60%蔗糖梯度柱的顶部缓慢加载1000个OD 260或200 OD 260的MBP(参见注释)。 MBP的产量远远低于FP,但是200 OD 260对于MBP轮廓分析是足够的。
    注意:小心不要打扰蔗糖的梯度。重要的是在加载过程中保持移液器吸头和梯度溶液的表面很好地接触(但不会突出到溶液中),否则可能形成液滴,并且梯度会受到干扰。
  15. 用SW55 Ti转子(Beckman Coulter)离心在237,020 x g,在4℃下离心1.5小时。
  16. 执行密度梯度分馏。分馏系统由注射泵,管穿孔架,检测器,馏分收集器和Peakchart软件(图2A)组成。梯度柱安装在管道穿孔机架上,并由支架底部的针刺穿(见视频2)。对于注射泵,将速度模式开关置于“正常”位置,并将流体方向切换到“关”位置;将控制模式旋钮转到“远程启动/停止”,并将流体速度调节至1.5 ml / min(图2B);对于UA-6检测器,将灵敏度值设置为“1”,将图表速度设置为“150 cm / h”(图2C)。该系统由Peakchart软件控制,核糖体分布由软件和检测器记录(图2D)。


    图2.密度梯度分馏系统。 A.分馏系统的概述。梯度柱连接到管道穿孔机架上,并由支架底部的针刺穿。梯度溶液通过注射泵中的追踪液从柱顶部缓慢推出,并通过UA-6检测器和Peakchart软件记录A254nm吸光度。 B.注射器泵的前面板。开关和旋钮的位置反映了分馏过程中的参数设置。 C. UA-6探测器的前面板。旋钮的位置反映了分馏过程中的参数设置。 D. Peakchart软件的截图。整个系统的启动或停止由底部中央的绿色按钮控制,配置文件以实时方式显示在屏幕上,并且程序完成后数据将自动保存。

    Video 2. Attachment of the gradient column to the density gradient fractionation system

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

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  17. 分析核糖体轮廓。典型的FP / MBP分布如图3所示。不同的峰分别表示40S小亚基,60S大亚基,80S单核细胞和多核糖体,良好的FP或MBP分离应显示具有这些不同峰和山的分布而不是多核糖体区域的下降曲线。注意,MBP的80S单体的峰值远低于FP,60S和80S级分通常以MBP组合。


    图3. FP和MBP的配置文件 FP(A)和MBP(B)通过超速离心以15-60%蔗糖梯度分离,然后通过梯度分馏系统分馏。 x轴表示相应梯度中的蔗糖浓度,y轴表示254nm处的吸光度水平。 40S:核糖体的小亚基; 60S:核糖体的大亚基; 80S:单体复合体

笔记

制备15-60%蔗糖梯度柱。除了用乙醇制备的CHX和CHL之外,所有储备溶液均用DEPC H 2 O制备。

  1. 制备不同蔗糖浓度的蔗糖溶液(对于10个梯度柱,表1):

    表1.制备15-60%蔗糖梯度柱的配方


  2. 将13 x 51 mm离心管(Beckman)放入可承受-80°C
    的机架中
  3. 从60%蔗糖层开始,将0.75ml 60%蔗糖溶液移至13×51mm离心管(Beckman)中,避免任何气泡,然后在-80℃下冷冻1小时。
  4. 将下一个梯度图层添加到表中所示的卷中,再次冻结,然后继续使用最后两层。
  5. 将蔗糖梯度柱储存在-80°C。梯度列可以在3个月内使用,如果它们被正确存储。
  6. 使用前,从冷冻箱中取出色谱柱,并在37℃的培养箱中解冻1小时,然后在冷藏室或冰箱中冷却1小时。

食谱

  1. 核糖体提取缓冲液
    0.2M Tris-HCl,pH8.5
    0.1 M KCI
    70 mM MgOAc
    50 mM EGTA
    0.25 M蔗糖 10 mM DTT
    50μg/ ml环己酰亚胺(CHX)(乙醇中50μg/μl)
    50μg/ ml氯霉素(CHL)(乙醇中50μg/μl)
    2.5 U / ml SUPERaseIN
  2. 蔗糖缓冲溶液
    0.4M Tris-HCl,pH9.0。
    0.2 M KCI
    0.005 M EGTA
    0.035M MgCl 2
    1.75 M蔗糖
    5 mM DTT
    50μg/ ml CHX(在乙醇中储存50μg/μl)
    50μg/ ml CHL(乙醇中50μg/μl)
  3. 再悬浮缓冲液
    0.2M Tris-HCl,pH9.0。
    0.2 M KCl
    0.025 M EGTA
    0.035M MgCl 2
    5 mM DTT
    50μg/ ml CHX(在乙醇中储存50μg/μl)
    50μg/ ml CHL(乙醇中50μg/μl)
  4. 10x蔗糖盐(15-60%蔗糖梯度柱)
    0.4M Tris-HCl,pH8.4
    0.2 M KCI
    0.1M MgCl 2

注意:如果不强调,所有缓冲液都用DEPC水制备;需要新增DTT,CHX,CHL和SUPERaseIN

致谢

这个协议是从我们以前的工作(Li等人,2016)改编而来的。感谢陈雪梅博士对本协议的建议。这项工作得到了深圳市科技创新委员会(JCYJ20151116155209176,KQCX2015033110464302,KY20150114)和深圳市重点实验室(ZDSYS20141118170111640)的资助。

参考

  1. de Jong,M.,van Breukelen,B.,Wittink,FR,Menke,FL,Weisbeek,PJ和Van den Ackerveken,G。(2006)。  拟南芥中的膜相关转录物;他们通过DNA微阵列分析和生物信息学进行分离和表征。植物J 46(4):708-721。
  2. Lerner,RS,Seiser,RM,Zheng,T.,Lager,PJ,Reedy,MC,Keene,JD and Nicchitta,CV(2003)。  在膜结合的核糖体上编码可溶性蛋白质的mRNA的分割和翻译。 9(9) :1123-1137。
  3. Li,S.,Le,B.,Ma,X.,Li,S.,You,C.,Yu,Y.,Zhang,B.,Liu,L.,Gao,L.,Shi, Zhao,Y.,Mo,B.,Cao,X. and Chen,X.(2016)。  在拟南芥中的膜结合多核糖体上的分阶段siRNA的生物发生 5。
  4. Li,S.,Liu,L.,Zhuang,X.,Yu,Y.,Liu,X.,Cui,X.,Ji,L.,Pan,Z.,Cao,X.,Mo, Zhang,F.,Raikhel,N.,Jiang,L. and Chen,X.(2013)。  MicroRNAs抑制拟南芥内质网上靶mRNA的翻译。 153(3):562 -574。
  5. Peter,W. and Johnson,AE(1994)。  信号序列识别和蛋白质靶向内质网膜。 Annu Rev Cell Biol 10:87-119。
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Copyright Zhao and Li. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhao, Y. and Li, S. (2017). Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings. Bio-protocol 7(15): e2436. DOI: 10.21769/BioProtoc.2436.
  2. Li, S., Le, B., Ma, X., Li, S., You, C., Yu, Y., Zhang, B., Liu, L., Gao, L., Shi, T., Zhao, Y., Mo, B., Cao, X. and Chen, X. (2016). Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5.
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