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本实验方案简略版
Jun 2018

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Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro
从兔肌肉组织中纯化球状肌动蛋白并利用芘荧光分析实验体外检测肌动蛋白的动态变化   

He SunHe Sun*Yuanyuan LuoYuanyuan Luo*Yansong MiaoYansong Miao  (*共同第一作者)
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Abstract

Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable and straightforward assays to study actin dynamics.

Keywords: Actin Purification (肌动蛋白纯化), Pyrene Assay (芘荧光检测), Actin Polymerization (肌动蛋白聚合), Actin Nucleation (肌动蛋白成核化), Formin (甲酸精)

Background

Actin dynamics play essential roles in crucial aspects of cellular biological functions, such as cell motility, endosomal trafficking, scaffolding and responses to external stimuli (Pollard and Cooper, 2009; Weinberg and Drubin, 2012; Sun et al., 2018). Rearrangements of actin cytoskeleton including nucleation, elongation, stabilization, capping, crosslinking and depolymerization are tightly controlled by actin-binding-proteins (ABPs) (Pollard, 2016). In vitro studies of the biochemical functions of those ABPs in actin regulation provides insights in understanding the mechanism of actin network regulation. Here, we provide an easy understanding protocol which can be applied for actin cytoskeleton-related biochemistry experiments, including actin purification, pyrene actin assay. The protocol provides approaches to monitor the actin assembly with other actin-binding-proteins in vitro and help researchers to learn about the actin dynamics process under different conditions. This protocol provides a clear table template for users to prepare a reaction mixture. Complicated calculation steps are simplified. This protocol uses high throughput multi-well methods instead of cuvette measurements to provide higher throughput and good parallel comparison among samples.

Materials and Reagents

  1. Pipette tips
  2. SnakeSkinTM Dialysis Tubing, 10K MWCO, 22 mm (Thermo Fisher, catalog number: 68100)
  3. Corning® Costar® 96-Well Cell Culture Plates (Sigma, catalog number: CLS3595)
  4. Greiner 96-well plates, polypropylene, black polypropylene wells flat bottom (Sigma, catalog number: M9685)
  5. HiPrepTM 16/60 Sephacryl S-300 HR (GE Healthcare, catalog number: 17116701)
  6. Nalgene Oak Ridge High-Speed PPCO Centrifuge Tubes (Thermo Scientific, catalog number: 3119-0050)
  7. Thinwall Polypropylene Tube (Beckman Coulter, catalog number: 326819)
  8. Bottle Assembly, Polycarbonate, for Rotor Type 45Ti (Beckman Coulter, catalog number: 355622)
  9. 6-384-well plates 
  10. Rabbit muscle acetone powder (Pel-Freez, USA, catalog number: 41995) (storage temperature: -20 °C or below)
  11. Pyrene-labeled actin (Cytoskeleton, lnc, catalog number: AP05-A) (storage temperature: -80 °C)
  12. Tris, ULTRA PURE (MP Biomedicals, catalog number: 4819623)
  13. ATP (MP Biomedicals, catalog number: 210000810)
  14. DTT (Gold Biotechnology, catalog number: DTT50)
  15. Calcium chloride (CaCl2) (Sigma, catalog number: 746495-100G)
  16. Potassium chloride (KCl) (Sigma, catalog number: P9541-1KG)
  17. Magnesium chloride (MgCl2) (Nacalai Tesque, catalog number: 20908-65)
  18. EGTA (VWR, catalog number: VWRC0732-100G)
  19. HCl (Sigma, catalog number: 320331)
  20. 100x G-buffer without CaCl2 (see Recipes)
  21. 1x G-buffer (see Recipes)
  22. 10x KME (see Recipes)
  23. F-buffer (see Recipes)

Equipment

  1. Pipettes
  2. Beaker (Schott-Duran, catalog number: SCOT211063604(E))
  3. Ultracentrifuge Optima XE (Beckman Coulter, model: OptimaTM XE)
  4. JA25.50 rotor (Beckman Coulter, model: JA-25.50)
  5. Ti50.2 rotor (Beckman Coulter, model: Type 50.2 Ti)
  6. Ti55 rotor (Beckman Coulter, model: SW 55 Ti)
  7. ÄKTApurifier (GE Healthcare, USA)
  8. Fluorescence spectrophotometer Cytation 5 (BioTek, USA)
  9. Mortar and pestle (Local market)
  10. Homogenizer (VWR, catalog number: VWRI432-0208)
  11. Spatula (Local Market)
  12. Ultrasonic Processor for Small and Medium Volume Applications (Sonics & Materials, INC. catalog number: VCX 750)
  13. Freezer

Software

  1. Origin software (Originlab Corporation, USA, https://www.originlab.com/)

Procedure

  1. Actin Purification
    Day 1:
    1. Prepare 1 L 1x G-buffer (see Recipes).
    2. Rehydrate and grind the 2.5 g lyophilized rabbit skeletal muscle acetone power in 50 ml G-buffer.
    3. Clear the solution by centrifugation at 27,000 x g for 1 h at 4 °C in the JA25.50 rotor (Beckman Coulter).
    4. Collect the solubilized actin in the supernatant.
    5. Polymerize actin in a 250 ml beaker by adding 50 mM KCl and 2 mM MgCl2 for 1 h followed by addition of 0.8 M KCl for 30 min at 4 °C.
    6. Pellet filamentous actin (F-actin) by centrifugation at 150,000 x g for 3 h at 4 °C using Ti50.2 rotor (Beckman Coulter).
    7. Pour off the supernatant and gently wash the surface of the pellet twice with 1 ml G-buffer.
    8. Transfer the pellet to a 10 ml homogenizer using a spatula (Do not let the pellet dry) and add 1 ml G-buffer to the homogenizer.
    9. Wash the centrifuge tube with G-buffer several times (use a total of 7 ml G-buffer). (This step allows the maximum recovery of actin).
    10. Homogenize the pellet by a homogenizer. 
    11. Brief sonicate the pellet using the Ultrasonic Processors on ice using 1 s on, 1 s off cycles at 30% amplitude for 4 s.
    12. Take out the homogenized solution and dialysis against G-buffer for 36 h at 4 °C using SnakeSkinTM dialysis tubing. Change the old G-buffer with a fresh one every 12 h (this step will allow the F-actin depolymerize).
    Day 2:
    1. Change the old G-buffer with the fresh buffer every 12 h.
    Day 3:
    1. Clean the monomeric actin by spinning at 200,000 x g for 2.5 h at 4 °C using a Ti55 rotor and collect the supernatant.
    2. Equilibrate the Sephacryl S-300 HR column with 150 ml 1x G-buffer.
    3. Load the supernatant collected from Step A14 to the column using 5 ml sample loop.
    4. Perform size exclusion chromatography using 1x G-buffer.
    5. Collect the monomeric actin peak after gel filtration chromatography (elution volume is around 50-60 ml) (Figure 1). 
    6. Determine the concentration of individual fractions from gel filtration and store the G-actin solution at 4 °C (The G-actin could be stored at 4 °C without significant activity lost).


      Figure 1. Typical gel filtration profile of G-actin purification. Actin was purified from Sephacryl S-300 HR column. G-actin was eluted at 50-60 ml. Inset showed SDS-PAGE analysis of G-actin eluted peak fraction.

  2. Actin Polymerization Assay
    1. Turn on the Fluorescence spectrophotometer Cytation 5 and start the software.
    2. Set up the reaction program:
      Parameter: excitation/emission: 365 nm/407 nm
      Time interval: 15 s
      Cycle number: 299
      Temperature: Room temperature
    3. Prepare 1 ml 10x KME buffer and 10 ml 1x G-buffer (see Recipes).
    4. Calculate the polymerization reaction materials needed based on Table 1. For one reaction, actin mix volume is 25 μl; maximum additional protein volume is 10 μl, 10x KME buffer volume is 12 μl and tops up with 1x G-buffer to total volume is 120 μl.
    5. Prepare target protein stock according to Table 1. 
    6. Prepare actin mix based on Table 2 and keep on ice for 5 min.
    7. Prepare reaction mix (without 10x KME) in Corning® Costar® 96-well cell culture plates according to Table 1.
    8. Add 12 μl 10x KME to each well together using a multichannel pipette and gently mix the reaction solution.
    9. Transfer 110 μl reaction solution into a 96-well black plate and initialize the Fluorescence spectrophotometer for data acquisition. 
    10. Plot the data using Origin software (Figure 1 is a typical example for actin polymerization assay).

      Table 1. Actin polymerization calculation example table

      *G-buffer is used to top up the reaction volume to 120 μl.
      **Maximum total volume of protein and protein buffer is 10 μl.

      Table 2. Actin mix calculation example table

      *Use the value of the actual G-actin and pyrene-labeled actin stocks employed for the experiments.
      **Volume is based on real reaction volume.


      Figure 2. Pyrene actin polymerization assay example. Two micromolar actin mixed with 5% pyrene-labeled actin was polymerized with indicated concentrations of Arabidopsis formin AtFH1(FH1COOH). Black, actin only; Blue, 200 nM AtFH1(FH1COOH); Purple, 500 nM AtFH1(FH1COOH). These three curves are the controls for the typical behavior of actin and formin.

Data analysis

All the in vitro pyrene actin assays were analyzed in Microsoft Excel or Origin software. Pyrene fluorescence intensity (Y-axis) is plotted as a function of time (X-axis). The final data could be plotted using the time window for the different purpose. To monitor the initial nucleation activity, usually < 1,000-1,500 s is sufficient to reflect the initial nucleation rate. The protein activities for different steps of actin polymerization may vary for some proteins depends on the protein stability during storage. Appropriate controls should be included in the same experiment (e.g., Actin only control should be included all the time to know whether actin is active). For non-stable proteins, the use of freshly prepared protein is recommended for biological replications.

Notes

  1. Pyrene actin assay results could be varied because of protein activity and practical reason. Replicate experiments are needed.
  2. Timing is important for the experiments. In order to get better reproducible results, time control of sample handling is necessary. For example, some actin-binding protein (such as formin) will rapidly accelerate actin polymerization in seconds. 
  3. Try to avoid creating any bubbles during pipetting.
  4. Use cut tips to pipet filamentous actin.
  5. Prepare fresh G-buffer and F-buffer for each day experiments.
  6. Fluorescence spectrophotometer Cytation 5 selection option:
    Monochromator-based Detection Modes (6-384-well plates):
    1. UV-Vis absorbance (230-999 nm, 1 nm increment)
    2. Fluorescence intensity (250-700 nm (850 nm option))
    3. Time resolved fluorescence (secondary)
    4. Luminescence 

Recipes

  1. 100x G-buffer without CaCl2 (10 ml)
    2 ml of 1 M Tris-HCl, pH 8
    1 ml of 0.2 M ATP, pH 7
    0.5 ml of 1 M DTT, pH 7
    Add ddH2O to 10 ml
    Make 110 μl aliquots and store at -20 °C
  2. 1x G-buffer (10 ml)
    100 μl 100x G-buffer
    9.9 ml cold ddH2O
    10 μl 0.1 M CaCl2
    Prepare fresh 1x G-buffer for one day’s experiment and store on ice
  3. 10x KME
    500 mM KCl
    10 mM MgCl2
    10 mM EGTA
    Store at room temperature
  4. F-buffer (10 ml)
    9 ml G-buffer
    1 ml 10x KME
    Prepare fresh F-buffer for one day’s experiment and store on ice

Acknowledgments

This study was supported by was NTU startup grant (M4081533), NIMBELS (NIM/01/2016), MOE Tier 2 (MOE2016-T2-1-005S), and MOE Tier 1 (RG38/17-S) to Y. Miao in Singapore. The protocol was adapted from Sun et al. (2018).

Competing interests

The authors declare no competing interests.

References

  1. Pollard, T. D. (2016). Actin and actin-binding proteins. Cold Spring Harb Perspect Biol 8(8) pii: a018226.
  2. Pollard, T. D. and Cooper, J. A. (2009). Actin, a central player in cell shape and movement. Science 326(5957): 1208-1212.
  3. Sun, H., Qiao, Z., Chua, K. P., Tursic, A., Liu, X., Gao, Y. G., Mu, Y., Hou, X. and Miao, Y. (2018). Profilin negatively regulates formin-mediated actin assembly to modulate PAMP-triggered plant immunity. Curr Biol 28(12): 1882-1895 e1887.
  4. Weinberg, J. and Drubin, D. G. (2012). Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol 22(1): 1-13.

简介

建立芘荧光测定法以监测动态肌动蛋白成核,伸长,加帽和体外拆解>。 该技术提供了简单的处理程序和直观的可视化数据分析。 通过在该方案中偶联肌动蛋白纯化和聚合测定,读者可以快速获得用于研究肌动蛋白动力学的经济且直接的测定。

【背景】肌动蛋白动力学在细胞生物学功能的关键方面起着重要作用,例如细胞运动,内体运输,支架和对外部刺激的反应(Pollard和Cooper,2009; Weinberg和Drubin,2012; Sun et al。 >,2018)。肌动蛋白细胞骨架的重排包括成核,伸长,稳定化,加帽,交联和解聚,受肌动蛋白结合蛋白(ABP)的严格控制(Pollard,2016)。 肌动蛋白调节中这些ABP的生化功能的体外>研究为理解肌动蛋白网络调节的机制提供了见解。在这里,我们提供了一个易于理解的协议,可用于肌动蛋白细胞骨架相关的生物化学实验,包括肌动蛋白纯化,芘肌动蛋白测定。该方案提供了用其他肌动蛋白结合蛋白体外监测肌动蛋白装配的方法,并帮助研究人员了解不同条件下的肌动蛋白动力学过程。该协议为用户提供了清晰的表格模板以制备反应混合物。简化了复杂的计算步骤。该协议使用高通量多孔方法代替比色皿测量,以提供更高的通量和样品之间的良好平行比较。

关键字:肌动蛋白纯化, 芘荧光检测, 肌动蛋白聚合, 肌动蛋白成核化, 甲酸精

材料和试剂

  1. 移液器吸头
  2. SnakeSkin TM 透析管,10K MWCO,22 mm(Thermo Fisher,目录号:68100)
  3. Corning ® Costar ® 96孔细胞培养板(Sigma,目录号:CLS3595)
  4. Greiner 96孔板,聚丙烯,黑色聚丙烯井平底(Sigma,目录号:M9685)
  5. HiPrep TM 16/60 Sephacryl S-300 HR(GE Healthcare,目录号:17116701)
  6. Nalgene Oak Ridge高速PPCO离心管(Thermo Scientific,目录号:3119-0050)
  7. 薄壁聚丙烯管(Beckman Coulter,目录号:326819)
  8. 瓶组件,聚碳酸酯,用于45Ti转子(Beckman Coulter,目录号:355622)
  9. 6-384孔板&nbsp;
  10. 兔肌肉丙酮粉(Pel-Freez,USA,目录号:41995)(储存温度:-20°C或以下)
  11. 芘标记的肌动蛋白(Cytoskeleton,Inc,目录号:AP05-A)(储存温度:-80°C)
  12. Tris,ULTRA PURE(MP Biomedicals,目录号:4819623)
  13. ATP(MP Biomedicals,目录号:210000810)
  14. DTT(黄金生物技术,目录号:DTT50)
  15. 氯化钙(CaCl 2 )(Sigma,目录号:746495-100G)
  16. 氯化钾(KCl)(Sigma,目录号:P9541-1KG)
  17. 氯化镁(MgCl 2 )(Nacalai Tesque,目录号:20908-65)
  18. EGTA(VWR,目录号:VWRC0732-100G)
  19. HCl(Sigma,目录号:320331)
  20. 没有CaCl 2 的100x G缓冲液(见食谱)
  21. 1x G缓冲液(见食谱)
  22. 10倍KME(见食谱)
  23. F缓冲区(见食谱)

设备

  1. 移液器
  2. 烧杯(Schott-Duran,目录号:SCOT211063604(E))
  3. 超速离心机Optima XE(Beckman Coulter,型号:Optima TM XE)
  4. JA25.50转子(Beckman Coulter,型号:JA-25.50)
  5. Ti50.2转子(Beckman Coulter,型号:50.2 Ti型)
  6. Ti55转子(Beckman Coulter,型号:SW 55 Ti)
  7. ÄKTApurifier(美国通用电气医疗集团)
  8. 荧光分光光度计Cytation 5(BioTek,USA)
  9. 砂浆和杵(当地市场)
  10. 均质器(VWR,目录号:VWRI432-0208)
  11. 抹刀(当地市场)
  12. 用于中小批量应用的超声波处理器(Sonics&amp; Materials,INC。目录号:VCX 750)
  13. 冰柜

软件

  1. Origin软件(Originlab Corporation,USA, https://www.originlab.com/ )

程序

  1. 肌动蛋白纯化
    第1天:
    1. 准备1 L 1x G缓冲液(参见配方)。
    2. 在50ml G-缓冲液中再水化并研磨2.5g冻干的兔骨骼肌丙酮粉末。
    3. 通过在JA25.50转子(Beckman Coulter)中在4℃下以27,000 x g >离心1小时来清除溶液。
    4. 将溶解的肌动蛋白收集在上清液中。
    5. 通过加入50mM KCl和2mM MgCl 2,在250ml烧杯中聚合肌动蛋白1小时,然后在4℃下加入0.8M KCl 30分钟。
    6. 通过使用Ti50.2转子(Beckman Coulter)在4℃下在150,000 离心3小时离心3小时来沉淀丝状肌动蛋白(F-肌动蛋白)。
    7. 倒出上清液,用1ml G缓冲液轻轻洗涤沉淀表面两次。
    8. 使用刮刀将颗粒转移到10ml匀浆器中(不要让颗粒干燥)并向均化器中加入1ml G-缓冲液。
    9. 用G缓冲液洗涤离心管数次(使用总共7ml G缓冲液)。 (此步骤可最大限度地恢复肌动蛋白)
    10. 用均化器将颗粒均化。&nbsp;
    11. 使用超声波处理器在冰上使用1秒开启,在30秒振幅下以1秒关闭循环4秒钟对颗粒进行超声处理。
    12. 取出均质溶液,使用SnakeSkin TM 透析管在4℃下用G缓冲液透析36小时。每12小时用新鲜的G缓冲液更换旧的G缓冲液(此步骤将允许F-肌动蛋白解聚)。
    第2天:
    1. 每12小时用新鲜缓冲液更换旧的G缓冲液。
    第3天:
    1. 使用Ti55转子在4℃下以200,000 x g >旋转2.5小时来清洁单体肌动蛋白,并收集上清液。
    2. 用150ml 1x G-缓冲液平衡Sephacryl S-300HR柱。
    3. 使用5ml样品环将从步骤A14收集的上清液加载到柱中。
    4. 使用1x G缓冲液进行尺寸排阻色谱。
    5. 凝胶过滤层析后收集单体肌动蛋白峰(洗脱体积约为50-60 ml)(图1)。&nbsp;
    6. 确定凝胶过滤中各个级分的浓度,并将G-肌动蛋白溶液储存在4°C(G-actin可以在4°C下储存而不会有明显的活性损失)。


      图1 G-肌动蛋白纯化的典型凝胶过滤曲线。 肌动蛋白从Sephacryl S-300 HR柱纯化。 G-肌动蛋白以50-60ml洗脱。插图显示了G-actin洗脱峰分数的SDS-PAGE分析。

  2. 肌动蛋白聚合分析
    1. 打开荧光分光光度计Cytation 5并启动软件。
    2. 设置反应计划:
      参数:激发/发射:365 nm / 407 nm
      时间间隔:15秒
      周期数:299
      温度:室温
    3. 准备1 ml 10x KME缓冲液和10 ml 1x G缓冲液(参见配方)。
    4. 根据表1计算所需的聚合反应物质。对于一个反应,肌动蛋白混合物体积为25μl;最大额外蛋白质体积为10μl,10x KME缓冲液体积为12μl,加满1x G缓冲液至总体积为120μl。
    5. 根据表1准备目标蛋白质原料。&nbsp;
    6. 根据表2制备肌动蛋白混合物并在冰上保持5分钟。
    7. 根据表1,在Corning ® Costar ® 96孔细胞培养板中制备反应混合物(不含10x KME)。
    8. 使用多通道移液管将12μl10xKME一起加入到每个孔中并轻轻混合反应溶液。
    9. 将110μl反应溶液转移到96孔黑色板中,初始化荧光分光光度计进行数据采集。&nbsp;
    10. 使用Origin软件绘制数据(图1是肌动蛋白聚合测定的典型实例)。

      表1.肌动蛋白聚合计算实例表

      * G缓冲液用于将反应体积补足至120μl。
      **蛋白质和蛋白质缓冲液的最大总体积为10μl。

      表2.肌动蛋白混合计算示例表

      *使用用于实验的实际G-肌动蛋白和芘标记的肌动蛋白原种的值。
      **体积基于实际反应量。


      图2.芘肌动蛋白聚合测定实施例。 将2微摩尔肌动蛋白与5%芘标记的肌动蛋白混合,用指定浓度的拟南芥formin AtFH1(FH1COOH)聚合。黑色,肌动蛋白;蓝色,200 nM AtFH1(FH1COOH);紫色,500nM AtFH1(FH1COOH)。这三条曲线是肌动蛋白和formin典型行为的对照。

数据分析

所有体外>芘肌动蛋白测定均在Microsoft Excel或Origin软件中进行分析。将芘荧光强度(Y轴)绘制为时间(X轴)的函数。可以使用时间窗口绘制最终数据以用于不同目的。为了监测初始成核活动,通常&lt; 1,000-1,500秒足以反映初始成核速率。对于一些蛋白质,肌动蛋白聚合的不同步骤的蛋白质活性可能不同,这取决于储存期间的蛋白质稳定性。适当的对照应包括在同一实验中(例如>,应始终包括肌动蛋白对照以了解肌动蛋白是否有效)。对于非稳定蛋白质,建议使用新鲜制备的蛋白质进行生物复制。

笔记

  1. 由于蛋白质活性和实际原因,芘肌动蛋白测定结果可以变化。需要重复实验。
  2. 时间对于实验很重要。为了获得更好的可重复结果,样品处理的时间控制是必要的。例如,一些肌动蛋白结合蛋白(如formin)会在几秒钟内迅速加速肌动蛋白聚合。&nbsp;
  3. 尽量避免在移液过程中产生任何气泡。
  4. 使用切割技巧来吸取丝状肌动蛋白。
  5. 为每天的实验准备新鲜的G缓冲液和F缓冲液。
  6. 荧光分光光度计Cytation 5选择选项:
    基于单色仪的检测模式(6-384孔板):
    1. 紫外 - 可见吸光度(230-999 nm,1 nm增量)
    2. 荧光强度(250-700 nm(850 nm选项))
    3. 时间分辨荧光(二级)
    4. 发光&NBSP;

食谱

  1. 100×G缓冲液,不含CaCl 2 (10 ml)
    2毫升1M Tris-HCl,pH8
    1毫升0.2 M ATP,pH 7
    0.5毫升1M DTT,pH 7
    将ddH 2 O加入10 ml
    制备110μl等分试样并储存在-20°C
  2. 1x G缓冲液(10 ml)
    100μl100xG缓冲液
    9.9毫升冷ddH 2 O
    10μl0.1MCaCl 2
    准备新鲜的1x G缓冲液进行一天的实验并储存在冰上
  3. 10倍KME
    500 mM KCl
    10mM MgCl 2
    10 mM EGTA
    在室温下储存
  4. F缓冲液(10毫升)
    9毫升G缓冲液
    1毫升10倍KME
    准备新鲜的F-缓冲液进行为期一天的实验并储存在冰上

致谢

这项研究得到了NTU启动资助(M4081533),NIMBELS(NIM / 01/2016),MOE Tier 2(MOE2016-T2-1-005S)和MOE Tier 1(RG38 / 17-S)的支持。在新加坡。该协议改编自Sun 等人>(2018)。

利益争夺

作者声明没有竞争利益。

参考

  1. Pollard,T。D.(2016)。 肌动蛋白和肌动蛋白结合蛋白。 Cold Spring Harb Perspect Biol > 8(8)pii:a018226。
  2. Pollard,T。D.和Cooper,J。A.(2009)。 肌动蛋白,细胞形态和运动的核心参与者。 科学> 326(5957):1208-1212。
  3. Sun,H.,Qiao,Z.,Chua,K.P.,Tursic,A.,Liu,X.,Gao,Y.G.,Mu,Y.,Hou,X。和Miao,Y。(2018)。 Profilin负调节formin介导的肌动蛋白装配,以调节PAMP引发的植物免疫。 Curr Biol > 28(12):1882-1895 e1887。
  4. Weinberg,J。和Drubin,D。G.(2012)。 网格蛋白介导的芽殖酵母内吞作用。 趋势细胞生物学 > 22(1):1-13。
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引用:Sun, H., Luo, Y. and Miao, Y. (2018). Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro. Bio-protocol 8(23): e3102. DOI: 10.21769/BioProtoc.3102.
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