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Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
富含精氨酸的肽可以主动介导液-液相分离   

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参见作者原研究论文

本实验方案简略版
Molecular Cell
Mar 2017

Abstract

Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).

Keywords: LLPS (LLPS), Amyotrophic lateral sclerosis (肌萎缩性侧索硬化), Peptides (肽), Arginine (精氨酸), Phase separation (相分离), Mass spec (质谱仪), C9orf72 (C9orf72), Stress granule (应激颗粒)

Background

Arginine-rich disordered domains are often found in RNA binding proteins, including the ones associated with neurodegenerative diseases (e.g., FUS, FMRP, hnRNPA1) (Varadi et al., 2015; Boeynaems et al., 2017). Also toxic arginine-rich repeat peptides (i.e., polyGR and polyPR) are produced in amyotrophic lateral sclerosis patients carrying the C9orf72 repeat expansion (Kwon et al., 2014; Mizielinska et al., 2014; Varadi et al., 2015; Boeynaems et al., 2016). While phase separation of uncharged low complexity domains had been studied before (Kato et al., 2012; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015), we developed this protocol to test whether arginine-rich domains could also contribute to phase separation. A schematic representation of the protocol can be seen in Figure 1.


Figure 1. Workflow of protein droplet formation and analysis. A diffuse arginine-rich peptide solution can be induced to undergo liquid-liquid phase separation by addition of RNA or molecular crowder (PEG). Resulting droplets can be further analyzed by fluorescence microscopy and FRAP.

Materials and Reagents

  1. Pipette tips
  2. Amicon® Ultra–0.5 ml centrifugal filters 3K (Merck, catalog number: UFC500396 )
  3. Cell counting slides (Bio-Rad Laboratories, catalog number: 1450015 )
  4. Clear adhesive tape
  5. Custom 20 to 60 amino acid peptides with free carboxy and amino-termini (Pepscan)
  6. Custom Alexa labeled RNA 30 ribonucleotide oligomers (IDT)
  7. MilliQ water
  8. Alexa Fluor® Protein Labeling Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A10235 )
  9. PEG300 (Sigma-Aldrich, catalog number: 81160 )
    Note: This product has been discontinued.
  10. Potassium phosphate monobasic (KH2PO4)
  11. Potassium phosphate dibasic (K2HPO4)
  12. Polyuridylic acid potassium salt (Sigma-Aldrich, catalog number: P9528 )
  13. Clear nail varnish
  14. 10x potassium buffer, pH 7 (see Recipes)

Equipment

  1. Pipette
  2. Bench-top centrifuge (Eppendorf, models: 5430 and 5810 R )
  3. trUView cuvettes (Bio-Rad Laboratories, catalog number: 1702510 )
    Note: This product has been discontinued.
  4. SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus )
  5. LSM 780 Meta NLO confocal microscope (ZEISS, model: LSM 780 NLO ) with 20x long range objective (ZEISS, model: LD Plan-Neofluor 20x/0.4 Corr Ph2 M27, catalog number: 421351-9970-000 )

Software

  1. Zen software (ZEISS)
  2. GraphPad Prism (GraphPad)
  3. Excel (Microsoft)

Procedure

Note: Dissolve aliquots of lyophilized custom peptides (Pepscan) in MilliQ water to a stock solution of 1 mM. Dissolve aliquots of lyophilized custom fluorescent RNA oligos (IDT) in MilliQ water to a stock solution of 100 µM. Dissolve lyophilized polyU potassium salt (Sigma-Aldrich) in MilliQ water to a stock solution of 10 µg/µl. Aliquots are stored at -20 °C.

  1. Fluorescent labeling of custom peptides
    1. Perform labeling reaction with desired Alexa Fluor® labeling kit according to the manufacturer’s instructions, but purify the peptide with the use of Amicon Ultra spin columns (minimum peptide size must be above 3 kDa).
    2. Add labeling reaction to spin column and centrifuge in bench top centrifuge for 10 min at 20,817 x g (rcf, max speed) at room temperature (Eppendorf 5430).
    3. Discard flow-through. Add 400 µl MilliQ water and centrifuge again. Repeat at least five times or until there is visually no more dye in the filtrate.
    4. Pipet remaining solution which still contains labeled peptide from spin column and adjust volume to obtain a 500 µM-1 mM stock solution. Concentration is calculated based on initial input.

  2. Induction of arginine-peptide phase separation by molecular crowder
    1. Mix by pipetting rigorously together the following reagents in MilliQ water to obtain described final concentrations.
      Note: Make sure to pipet PEG300 slowly and always with a new pipet tip to prevent errors due to the high viscosity of the solution.
      Potassium buffer (10x stock) to 1x (see Recipes)
      PEG300 (100%) to 30%
      Peptide (1 mM) to desired concentration (1-250 µM)
    2. If the peptide is prone to phase separation the solution should turn cloudy or opaque instantaneously at room temperature. For example, see Figure 2A. Briefly cooling on ice can promote phase separation of proteins that phase separate only weakly.
    3. Quantify extent of phase separation by measuring OD600 of 60 µl samples using trUView microcuvettes in a SmartSpec Plus (or similar) spectrophotometer.


      Figure 2. Examples of peptides and droplet formation. A. Peptide sequence and arginine content affect phase transition; Arginines highlighted in green. B. RNA and arginine-rich peptides colocalize upon phase separation, as seen by fluorescence microscopy.

  3. Induction of arginine-peptide phase separation by RNA
    1. Mix by pipetting rigorously together the following reagents in MilliQ water to obtain described final concentrations:
      Potassium buffer (10x stock) to 1x
      PolyU potassium salt (10 µg/µl) to 1 µg/µl
      Peptide (1 mM) to desired concentration (1-250 µM)
    2. If the peptide is prone to phase separation the solution should turn cloudy or opaque instantaneously at room temperature.
    3. Quantify extent of phase separation by measuring OD600 of 60 µl samples using trUView microcuvettes in a SmartSpec Plus (or similar) spectrophotometer.

  4. Imaging arginine-peptide phase separation by fluorescence microscopy
    1. Prepare phase separated peptide solution as described above. Spike the sample with fluorescent peptide or RNA oligomer to reach 200 nM and 100 nM respectively as final concentrations.
    2. Pipet 20 µl of the sample into each chamber of the cell counting slides (see Figure 3).
    3. Seal the chambers using clear adhesive tape and cover with nail varnish to prevent evaporation during imaging (see Figure 3).
    4. Let the samples equilibrate at room temperature in the dark for at least 30 min.
    5. Image samples on a LSM 780 Meta NLO confocal microscope with 20x long range objective. For example, see Figure 2B.


      Figure 3. Workflow of microscope chamber preparation. A solution containing a blue dye was used for illustration. Step 1: Pipet sample in the incubation chamber; Step 2: Seal both sides of the chamber with clear adhesive tape; Step 3: Apply clear nail varnish to make chamber securely air tight.

  5. Fluorescence recovery after photobleaching (FRAP) analysis
    1. Samples are prepared as described above.
    2. Bleach a circular area of 1-2 µM radius at 100% laser power in the center of a droplet with a radius between 5 µM and 10 µM. Use of larger droplets is advised so bleached area does not cover whole droplet compromising the study of intradroplet diffusion. When droplets do not reach this size spontaneously, incubation chambers were briefly centrifuged for 30 sec at 201 x g (rcf; 1,000 rpm on Eppendorf 5810 R centrifuge).
    3. Monitor fluorescence recovery after bleaching for at least 60 sec using Zen software (Figure 4A).
    4. Make sure to record simultaneously the fluorescence intensity over time of the background solution and an unbleached reference droplet.
    5. Export raw data to Microsoft Excel for analysis.

Data analysis

FRAP data analysis using Microsoft Excel and Prism (Figure 4B).

  1. Import the raw data from the Zen software into Microsoft Excel.
  2. Subtract the fluorescence intensity of the background from the bleached droplet and reference droplet.
  3. Subtract the fluorescence intensity of the first time point post-bleach from the bleached droplet. This step will normalize the first post-bleach time point to zero.
  4. Divide the fluorescence intensities of the bleached droplet by the pre-bleach fluorescence intensity. Do this as well for the reference droplet. This will normalize the pre-bleach time point to 1.
  5. Divide the fluorescence intensities of the bleached droplet by the fluorescence intensities of the reference droplet. This will correct for photobleaching due to prolonged exposure during the time course experiment.
  6. Import the normalized data into GraphPad Prism.
  7. Plot the fluorescence intensities as a function of time.
  8. Average FRAP curves of at least 15 droplets and evaluate differences between multiple conditions using repeated measures ANOVA.


    Figure 4. FRAP analysis of fluorescent protein droplets. A. Example time series of a FRAP measurement. The fluorescent PR signal was bleached inside the droplet and recovery was followed over time. B. Overview of raw data normalization. Step 1: Raw data as exported from Zen software; Step 2: Data after background normalization; Step 3: Data after bleach normalization; Step 4 + 5: Final data after fractional normalization and spontaneous bleach correction. Step numbers correspond to numbers in the text (Data analysis).

Recipes

  1. 10x potassium buffer
    1. Make 1 M stock solutions of K2HPO4 and KH2PO4 in MilliQ water
    2. Combine 61.5 ml of 1 M K2HPO4 with 38.5 ml of 1 M KH2PO4 to 100 ml 10x potassium buffer

Acknowledgments

This protocol was described in brief in Boeynaems et al. (2017). Research was funded by the KU Leuven, VIB, the European Research Council in the context of the European’s Seventh Framework Programme (FP7/2007-2013 and ERC grant agreement No. 340429), the Research Foundation Flanders (FWO) G.0983.14N, the Interuniversity Attraction Poles Programme P7/16 initiated by the Belgian Science Policy Office, the Association Belge contre les Maladies Neuro-Musculaires (ABMM), the ALS Liga (Belgium) and the ‘Opening the Future’ Fund. S.B. received a PhD fellowship from the Agency for Innovation by Science and Technology (IWT). P.T. was supported by the Odysseus grant G.0029.12 from Research Foundation Flanders (FWO).

References

  1. Boeynaems, S., Bogaert, E., Kovacs, D., Konijnenberg, A., Timmerman, E., Volkov, A., Guharoy, M., De Decker, M., Jaspers, T., Ryan, V. H., Janke, A. M., Baatsen, P., Vercruysse, T., Kolaitis, R. M., Daelemans, D., Taylor, J. P., Kedersha, N., Anderson, P., Impens, F., Sobott, F., Schymkowitz, J., Rousseau, F., Fawzi, N. L., Robberecht, W., Van Damme, P., Tompa, P. and Van Den Bosch, L. (2017). Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics. Mol Cell 65(6): 1044-1055 e1045.
  2. Boeynaems, S., Bogaert, E., Michiels, E., Gijselinck, I., Sieben, A., Jovicic, A., De Baets, G., Scheveneels, W., Steyaert, J., Cuijt, I., Verstrepen, K. J., Callaerts, P., Rousseau, F., Schymkowitz, J., Cruts, M., Van Broeckhoven, C., Van Damme, P., Gitler, A. D., Robberecht, W. and Van Den Bosch, L. (2016). Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Sci Rep 6: 20877.
  3. Burke, K. A., Janke, A. M., Rhine, C. L. and Fawzi, N. L. (2015). Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol Cell 60(2): 231-241.
  4. Jovicic, A., Mertens, J., Boeynaems, S., Bogaert, E., Chai, N., Yamada, S. B., Paul, J. W., 3rd, Sun, S., Herdy, J. R., Bieri, G., Kramer, N. J., Gage, F. H., Van Den Bosch, L., Robberecht, W. and Gitler, A. D. (2015). Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18(9): 1226-1229.
  5. Kato, M., Han, T. W., Xie, S., Shi, K., Du, X., Wu, L. C., Mirzaei, H., Goldsmith, E. J., Longgood, J., Pei, J., Grishin, N. V., Frantz, D. E., Schneider, J. W., Chen, S., Li, L., Sawaya, M. R., Eisenberg, D., Tycko, R. and McKnight, S. L. (2012). Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149(4): 753-767.
  6. Kwon, I., Xiang, S., Kato, M., Wu, L., Theodoropoulos, P., Wang, T., Kim, J., Yun, J., Xie, Y. and McKnight, S. L. (2014). Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345(6201): 1139-1145.
  7. Lin, Y., Protter, D. S., Rosen, M. K. and Parker, R. (2015). Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol Cell 60: 208-219.
  8. Mizielinska, S., Gronke, S., Niccoli, T., Ridler, C. E., Clayton, E. L., Devoy, A., Moens, T., Norona, F. E., Woollacott, I. O., Pietrzyk, J., Cleverley, K., Nicoll, A. J., Pickering-Brown, S., Dols, J., Cabecinha, M., Hendrich, O., Fratta, P., Fisher, E. M., Partridge, L. and Isaacs, A. M. (2014). C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345(6201): 1192-1194.
  9. Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A. P., Kim, H. J., Mittag, T. and Taylor, J. P. (2015). Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163(1): 123-133.
  10. Patel, A., Lee, H. O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M. Y., Stoynov, S., Mahamid, J., Saha, S., Franzmann, T. M., Pozniakovski, A., Poser, I., Maghelli, N., Royer, L. A., Weigert, M., Myers, E. W., Grill, S., Drechsel, D., Hyman, A. A. and Alberti, S. (2015). A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162(5): 1066-1077.
  11. Varadi, M., Zsolyomi, F., Guharoy, M. and Tompa, P. (2015). Functional advantages of conserved intrinsic disorder in RNA-binding proteins. PLoS One 10(10): e0139731.

简介

研究蛋白质的液 - 液相分离(LLPS)提供了无膜细胞器的生物发生和疾病中病理蛋白聚集的关键见解。 我们已经建立了诱导富含精氨酸的肽的相分离的方案,这允许研究其分子决定因素和动力学(Boeynaems等,2017)。
【背景】富含精氨酸的无序结构域通常存在于RNA结合蛋白中,包括与神经变性疾病相关的蛋白(例如FUS,FMRP,hnRNPA1)(Varadi等人,2015; Boeynaems等,2017)。 在携带C9orf72重复扩增的肌萎缩性侧索硬化患者中也产生有毒的富含精氨酸的重复肽(即polyGR和polyPR)(Kwon等,2014; Mizielinska等,2014; Varadi等,2015; Boeynaems et 等等,2016)。 (Kato et al。,2012; Burke et al。,2015; Lin et al。,2015; Molliex et al。,2015; Patel et al。,2015),虽然已经研究了不带电的低复杂度域的相分离, 开发了该方案来测试富含精氨酸的结构域是否也有助于相分离。 协议的示意图可以在图1中看到。

关键字:LLPS, 肌萎缩性侧索硬化, 肽, 精氨酸, 相分离, 质谱仪, C9orf72, 应激颗粒

材料和试剂

  1. 移液器提示
  2. 超薄0.5ml离心过滤器3K(默克,目录号:UFC500396)
  3. 细胞计数载玻片(Bio-Rad Laboratories,目录号:1450015)
  4. 清除胶带
  5. 定制20至60个具有游离羧基和氨基末端的氨基酸肽(Pepscan)
  6. 自定义Alexa标记的RNA 30核糖核苷酸寡聚体(IDT)
  7. MilliQ水
  8. Alexa Fluor蛋白标记试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:A10235)
  9. PEG300(Sigma-Aldrich,目录号:81160)
    注意:本产品已停产。
  10. 磷酸二氢钾(KH 2 PO 4)
  11. 磷酸二氢钾(K 2/2 HPO 4)
  12. 聚尿苷酸钾盐(Sigma-Aldrich,目录号:P9528)
  13. 清除指甲油
  14. 10倍钾缓冲液,pH 7(参见食谱)

设备

  1. 移液器
  2. 台式离心机(Eppendorf,型号:5430和5810 R)
  3. trUView比色皿(Bio-Rad Laboratories,目录号:1702510)
    注意:本产品已停产。
  4. SmartSpec Plus分光光度计(Bio-Rad Laboratories,型号:SmartSpec Plus)
  5. 具有20倍长距离目标(ZEISS,型号:LD Plan-Neofluor 20x / 0.4 Corr Ph2 M27,目录号:421351-9970-000)的LSM 780 Meta NLO共聚焦显微镜(ZEISS,型号:LSM 780 NLO)

软件

  1. 禅宗软件(ZEISS)
  2. GraphPad Prism(GraphPad)
  3. Excel(Microsoft)

程序

注意:将MilliQ水中冻干定制肽(Pepscan)的等分试样溶解到1 mM的储备溶液中。将MilliQ水中冻干的定制荧光RNA寡核苷酸(IDT)的等分试样溶解至100μM的储备溶液。将溶解在MilliQ水中的冻干的多尿素钾(Sigma-Aldrich)溶解到10μg/μl的储备溶液中。等分试样储存在-20°C

  1. 定制肽的荧光标记
    1. 根据制造商的说明书,使用所需的Alexa Fluor标签试剂盒进行标记反应,但使用Amicon Ultra自旋柱纯化肽(最小肽大小必须高于3 kDa)。
    2. 在室温下(Eppendorf 5430),以20,817 x g(rcf,最大速度)在台式离心机中加入标记反应并离心10分钟。
    3. 丢弃流通。加入400μlMilliQ水,再次离心。重复至少五次,或直到滤液中不再有染料。
    4. 吸管剩余溶液,其仍含有来自旋转柱的标记肽,并调节体积以获得500μM-1mM储备溶液。浓度根据初始投入计算。

  2. 通过分子挤压诱导精氨酸 - 肽相分离
    1. 通过在MilliQ水中将以下试剂严格移液混合以获得所述最终浓度。
      注意:请务必缓慢移液PEG300,并始终使用新的吸头,以防止由于溶液的高粘度引起的错误。
      钾缓冲液(10x储备)至1x(参见食谱)
      PEG300(100%)至30%
      肽(1mM)至所需浓度(1-250μM)
    2. 如果肽易于相分离,则溶液在室温下瞬间变为浑浊或不透明。例如参见图2A。在冰上简单冷却可以促进相分离的蛋白质的相分离
    3. 通过使用SmartSpec Plus(或类似)分光光度计中的trUView微量滴定板测量60μl样品的OD 600,来量化相分离程度。


      图2.肽和液滴形成的实例。A.肽序列和精氨酸含量影响相变;精氨酸以绿色突出显示。 B.通过荧光显微镜观察,RNA和富含精氨酸的肽在相分离时共定位
  3. 通过RNA诱导精氨酸 - 肽相分离
    1. 通过在MilliQ水中将以下试剂严格移液混合,以获得所述最终浓度:
      钾缓冲液(10x储备)至1x
      PolyU钾盐(10μg/μl)至1μg/μl
      肽(1mM)至所需浓度(1-250μM)
    2. 如果肽易于相分离,则溶液在室温下瞬间变为浑浊或不透明。
    3. 通过使用SmartSpec Plus(或类似)分光光度计中的trUView微量滴定板测量60μl样品的OD 600,来量化相分离程度。

  4. 通过荧光显微镜成像精氨酸 - 肽相分离
    1. 如上所述制备相分离的肽溶液。用荧光肽或RNA寡聚体刺激样品,分别达到200nM和100nM作为终浓度
    2. 将20μl样品吸入细胞计数载玻片的每个腔室(参见图3)。
    3. 使用透明胶带和盖子用指甲油密封腔室,以防止成像期间蒸发(见图3)
    4. 让样品在室温下在黑暗中平衡至少30分钟
    5. 在LSM 780 Meta NLO共聚焦显微镜上具有20倍长距离目标的图像样本。例如参见图2B。


      图3.显微镜室制备的工作流程使用含有蓝色染料的溶液进行说明。步骤1:在孵育室中吸取样品;步骤2:用透明胶带密封室的两侧;步骤3:使用清漆指甲油使得室内气密性牢固。

  5. 光漂白后的荧光恢复(FRAP)分析
    1. 样品如上所述制备。
    2. 在半径为5μM至10μM的液滴的中心,以100%的激光功率漂白半径为1-2微米的圆形区域。建议使用较大的液滴,因此漂白区域不会覆盖整个液滴,从而影响了液滴内扩散的研究。当液滴自发地达不到这个大小时,孵化室在201 x g(rcf; 1000 rpm,Eppendorf 5810 R离心机)上短暂离心30秒。
    3. 使用Zen软件在漂白至少60秒后监测荧光恢复(图4A)。
    4. 确保同时记录背景溶液和未漂白参考液滴的荧光强度。
    5. 将原始数据导出到Microsoft Excel以进行分析。

数据分析

使用Microsoft Excel和Prism进行FRAP数据分析(图4B)。

  1. 将原始数据从Zen软件导入Microsoft Excel。
  2. 从漂白的液滴和参比液滴中减去背景的荧光强度
  3. 从漂白的液滴中减去漂白后第一个时间点的荧光强度。此步骤将第一个漂白后时间点归一化为零。
  4. 用漂白后的荧光强度除去漂白液滴的荧光强度。对于参考液滴也是这样做的。这将使漂白前的时间点正常化为1.
  5. 用参考液滴的荧光强度除去漂白液滴的荧光强度。这将在时间过程实验期间由于长时间曝光而校正光漂白。
  6. 将归一化数据导入GraphPad Prism。
  7. 绘制荧光强度作为时间的函数
  8. 至少15个液滴的平均FRAP曲线,并使用重复测量方差分析评估多个条件之间的差异

    图4.荧光蛋白液滴的FRAP分析。A. FRAP测量的时间序列示例。荧光PR信号在液滴内漂白,随着时间的推移恢复。 B.原始数据归一化概述。步骤1:从禅软件导出的原始数据;步骤2:背景规范化后的数据;步骤3:漂白标准化后的数据;步骤4 + 5:分数归一化和自发漂白校正后的最终数据。步数对应于文本中的数字(数据分析)。

食谱

  1. 10倍钾缓冲液
    1. 在MilliQ水中制备1 K K 2 2 HPO 4和KH 2 PO 4的储备溶液
    2. 将61.5ml 1KH 2 HPO 4+与38.5ml 1M KH 2 PO 4·4结合至100ml 10倍钾缓冲液

致谢

这个协议在Boeynaems等人(2017)中简要描述。研究由欧洲研究委员会(KU Leuven),欧洲研究理事会(VIB),欧洲第七框架计划(FP7 / 2007-2013和ERC拨款协议第340429号),研究基金会佛兰德(FWO)G.0983.14N,比利时科学政策办公室发起的"跨学院吸引力波兰计划"P7 / 16,马拉蒂斯神经肌肉协会(ABMM),ALS Liga(比利时)和"开放未来"基金发起。 S·B·获得了科技创新机构(IWT)的博士研究生奖学金。公厕得到了研究基金会法兰德斯(FWO)的Odysseus G.0029.12补助。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Boeynaems, S., De Decker, M., Tompa, P. and Van Den Bosch, L. (2017). Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation. Bio-protocol 7(17): e2525. DOI: 10.21769/BioProtoc.2525.
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