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Binding Assay of Cytosolic Proteins to the Cytoskeleton
胞质蛋白与细胞骨架相互作用研究   

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Abstract

Many cellular proteins interact with the cytoskeleton (both actin filaments and microtubules), either dynamically or permanently. This interaction is required during different aspects of the cell life, for example during the process of cell division. In addition, many enzymes interact transiently with actin filaments and microtubules in order to promote their cellular distribution. Several substances with inhibitory capacity can affect this binding and cause damages to cells. This protocol allows to analyze whether a protein interacts with either actin filaments or microtubules and, when applicable, the conditions controlling this interaction.
The test is based on the specific binding between the protein of interest and the cytoskeletal filaments. As shown schematically in the diagram of example (see below), the test starts from the cell lysate to which actin filaments (produced from monomeric actin) are added. The mixture (performed under different experimental conditions chosen by the operator) is then incubated so that the protein of interest (in the example, myosin) binds to actin filaments. The sample is then centrifuged in order to separate unbound or weakly-bound proteins from actin filaments to which both the protein of interest and, eventually, traces of less specific proteins are associated.

Keywords: Actin filament(肌动蛋白丝), Microtubule(微管), Myosin(肌球蛋白), Protein binding(蛋白结合)


Materials and Reagents

  1. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (≥ 97.0%) (Sigma-Aldrich, catalog number: E3889 )
  2. Guanosine 5’-triphosphate sodium salt hydrate (GTP) (≥ 95%) (Sigma-Aldrich, catalog number: G8877 )
  3. DL-Dithiothreitol (DTT) (≥ 99.0%) (Sigma-Aldrich, catalog number: 43819 )
  4. Adenosine 5’-triphosphate magnesium salt (ATP) (95-98%) (Sigma-Aldrich, catalog number: A0770 )
  5. PIPES disodium salt (≥ 99%) (Sigma-Aldrich, catalog number: P3768 )
  6. Glycerol (≥ 99%) (Sigma-Aldrich, catalog number: G5516 )
  7. MgCl2 (≥ 98%) (anhydrous) (Sigma-Aldrich, catalog number: M8266 )
  8. Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S9378 )
  9. Taxol (Paclitaxel from Taxus brevifolia) (≥ 95%) (Sigma-Aldrich, catalog number: T7402 )
  10. Bovine Tubulin (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: TL238-B )
  11. Actin protein from rabbit skeletal muscle (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: AKL99 )
  12. 2D Quant Kit (General Electric Company) or Bradford Protein Assay kit (Bio-Rad Laboratories)
  13. Tubulin Dilution Buffer (TDB) (see Recipes)
  14. Glycerol buffer (GB) (see Recipes)
  15. “Sucrose cushion” buffer (SC) (see Recipes)
  16. General actin buffer (A-buffer) (see Recipes)
  17. Polymerization Inducer (PI) (see Recipes)
  18. Cushion buffer for F-actin (see Recipes)

Equipment

  1. Apparatus for the lysis of cells and tissues (the type of apparatus for cell lysis depends on the cellular system with which one is working; animal cells can be lysed easily while plant cells, because of the presence of the cell wall, require more energetic methods such as freezing under liquid nitrogen and powdering with pestle and mortar. It is therefore not possible to give precise indications but we prefer to leave the choice to individual operators.)
  2. Electrophoretic apparatus (Mini-PROTEAN® II Electrophoresis Cell) (Bio-Rad Laboratories)
  3. Ultracentrifuge (Beckman Coulter, model: Optima LE-80 K) equipped with a 70 Ti fixed-angle rotor and adapters for 4-ml polyallomer tubes
  4. Bio-Rad Mini-Trans-Blot Cell Apparatus (optional)

Software

  1. ImageJ or Bio-Rad Quantity One

Procedure

  1. Microtubule binding assay
    1. Preparation of the soluble (cytoplasmic) extract
      1. This step necessarily depends on the cells or tissues on which one is working. In theory, soft tissues (such as animal tissues) can be lysed directly in the lysis buffer using either plastic Eppendorf pestles or an Ultra-Turrax homogenizer in case of complex tissues. For plant cells, it is preferable to freeze the tissue of interest under liquid nitrogen and then grind it with pestle and mortar. After the tissue was completely powdered, the liquid nitrogen is allowed to evaporate. The tissue powder is moved to a suitable tube, like eppendorf tubes or 15-ml conical tubes (depending on the amount of starting material). The lysis buffer is added and the sample is incubated at 4 °C or on ice for 15 min with gentle agitation. At the end, the sample is centrifuged at high speed (> 100,000 x g) for pelleting cellular debris and for getting cytosolic proteins in the supernatant. Although the extract of soluble proteins can be frozen under liquid nitrogen and stored at -80 °C, we always prefer to work with fresh material because it is difficult to estimate the temporal stability of the extract. The composition of lysis buffer may be adjusted from species to species, please refer to the literature on the specific tissue in order to determine the best lysis buffer.
      2. Determine protein concentration of the cytoplasmic extract in order to mix known protein quantities. Several commercial kits are available for determination of protein concentration (such as the 2D Quant Kit or the Bradford Protein Assay kit). Always take an aliquot for electrophoresis.
    2. Preparation of microtubules
      1. Thaw one aliquot of lyophilized tubulin (animal or plant source, 1 mg), then add 200 μl of TDB so that the final concentration is 5 mg/ml.
      2. Add 25 μl of GB and incubate at 35 °C for 20 min.
      3. Mix 1.8 ml of TDB with 81 μl of 500 μM taxol (TDB-T). Incubate at 35 °C for 15-20 min in such a way that the temperature of the taxol solution is the same of tubulin.
      4. At the end of incubation, add 1.8 ml of TDB-T to 225 μl of the tubulin sample. Mix gently. Tubulin is now in a final volume of ≈ 2 ml at a concentration of 0.5 mg/ml.
      5. Prepare additional TDB-T by mixing 1.8 ml of TDB with 81 μl of 500 μM taxol.
    3. Preparing the binding mix.
      Mix the cytosolic extract (in variable amounts, for example from 0 to 40 microliters, in order to work with a range of 10-100 micrograms of protein) with a constant volume of the microtubule sample (40 microliters). Use the remaining volume for the addition of other substances (i.e. inhibitors), and then adjust the volume to 100 microliters with TDB-T buffer. Volumes can be increased correspondingly to accommodate for different rotors and tubes.
    4. Incubate the samples at room temperature for 30 min.
      The test temperature can be adjusted if it is a variable examined in the experiment.
    5. Separation of cytoskeletal filaments
      1. Centrifuge the samples at 100,000 x g for 40 min at 20 °C over 0.5 ml of sucrose cushion buffer.
      2. After centrifugation, the supernatant is located above the sucrose cushion and may therefore be removed with a pipette without touching the sucrose cushion. Process the supernatants for electrophoresis.
      3. Resuspend the pellets directly into a denaturing buffer suitable for electrophoretic analysis (e.g. Laemmli buffer) (max. 100 microliters).
    6. Analysis
      Test individual fractions (supernatants and pellets) by electrophoresis (SDS-PAGE) and possibly by western blot, the latter being required to identify specifically and to quantify the protein of interest.

  2. Actin filament binding assay
    1. Preparation of cell lysate
      1. It depends on the particular tissue or cells on which one is working. See the previous protocol for more details.
      2. Determine the protein concentration of samples using commercial kits (such as the 2D Quant Kit or the Bradford Protein Assay kit) and always collect one aliquot for electrophoresis.
    2. Preparation of actin filaments
      1. Thaw one aliquot of lyophilized actin (animal or plant sources, 1 mg).
      2. Dilute the actin to a concentration of 10 mg/ml by adding 100 μl of A-buffer. Remove solution from the tube (usually an Eppendorf tube) and place in 5-ml or 15-ml tubes.
      3. Dilute the actin at a concentration of 0.4 mg/ml with A-buffer (100 μl of actin + 2.4 ml of A-buffer). Incubate on ice for 1 h (agitation is not required).
      4. Add the Polymerization Inducer 1x final: 2.25 ml + 0.25 ml of actin Polymerization Inducer 10x. Incubate at room temperature for 1 h.
    3. Preparing the binding mix.
      Mix the cytosolic extract (in variable amounts, for example from 0 to 40 microliters, in order to work with a range of 10-100 micrograms of protein) with a constant volume of the actin filament sample (50 microliters). Use the remaining volume for the addition of other substances (i.e. inhibitors), and then adjust the volume to 100 microliters with the A-buffer/Polymerization Inducer mix (which is the same buffer of the actin solution). Volumes can be increased correspondingly to accommodate for different rotors and tubes.
    4. Incubate samples at room temperature for 30-60 min.
      The test temperature can be adjusted if it is a variable examined in the experiment.
    5. Separation of cytoskeletal filaments
      1. Centrifuge samples at 150,000 x g for 60-90 min at 20 °C over 0.5 ml of the cushion buffer for F-actin.
      2. Take the supernatants and process them for electrophoresis.
      3. Resuspend the pellets in 100 μl of a denaturing buffer suitable for electrophoretic analysis (e.g. Laemmli buffer).
    6. Analysis
      1. Test individual fractions (supernatants and pellets) by electrophoresis (SDS-PAGE) and possibly by western blot, the latter being required to identify specifically and to quantify the protein of interest (Figure 1).
      2. In both cases, the relative quantification of bands after western blot analysis can be performed using image acquisition systems and specific software (free like ImageJ or paid such as the Bio-Rad Quantity One).


        Figure 1. Example of binding analysis with actin filaments. Specifically, the protein myosin from rabbit skeletal muscle (arrow) was incubated with filamentous actin (arrowhead). S and P indicate the supernatants and pellets, respectively. Lane 1, molecular weight standard, the value of which is indicated by numbers on the left. Lane 2, myosin (commercially available). Lanes 3-4, supernatant and pellet obtained after incubation of myosin with actin filaments. Lanes 5-6, supernatant and pellet obtained after incubation of myosin with actin filaments in the presence of 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP, a non-hydrolyzable analogue of ATP). Lanes 7-8, supernatant and pellet obtained after incubation of myosin with actin filaments in the presence of ATP. Note that myosin binds weakly in the presence of ATP but very strongly in the presence of AMPPNP.

Recipes

  1. Tubulin Dilution Buffer (TDB)
    80 mM Pipes, pH 6.8
    1 mM EGTA
    1 mM MgCl2
    2 mM GTP
  2. Glycerol buffer (GB)
    80 mM Pipes, pH 6.8
    1 mM EGTA
    1 mM MgCl2
    60% (w/v) glycerol
  3. “Sucrose cushion” buffer (SC)
    80 mM Pipes, pH 6.8
    1 mM EGTA
    1 mM MgCl2
    10% (w/v) sucrose
    20 μM taxol
  4. General actin buffer (A-buffer)
    5 mM Tris-HCl pH 8.0
    0.2 mM CaCl2
    0.2 mM ATP
    0.5 mM DTT
  5. 10x Polymerization Inducer (PI)
    500 mM KCl
    20 mM MgCl2
    10 mM ATP
  6. Cushion buffer for F-actin
    5 mM Tris-HCI pH 8.0
    2 mM MgCl2
    50 mM KCl
    10% (v/v) glycerol

Acknowledgments

The protocol was adapted from a previously published paper: Del Duca et al. (2013). The work was supported by PRIN 2007 (grant no. 2007RZCW5S_003) and PRIN 2008 (grant no. 2008BK7RXB), funded by the Italian Ministry of University and Research, and by Bologna University (RFO 2010 [grant no. RFO10DELDU] and RFO 2011 [grant no. RFO11DELDU]) to S.D.D.

References

  1. Cai, G., Faleri, C., Del Casino, C., Emons, A. M. and Cresti, M. (2011). Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules. Plant Physiol 155(3): 1169-1190.
  2. Del Duca, S., Faleri, C., Iorio, R. A., Cresti, M., Serafini-Fracassini, D. and Cai, G. (2013). Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics. Plant Physiol 161(4): 1706-1721. 
  3. Del Duca, S., Serafini-Fracassini, D., Bonner, P., Cresti, M. and Cai, G. (2009). Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments. Biochem J 418(3): 651-664.

简介

许多细胞蛋白质与细胞骨架(肌动蛋白丝和微管)相互作用,动态或永久。这种相互作用在细胞生命的不同方面是需要的,例如在细胞分裂过程中。此外,许多酶与肌动蛋白丝和微管瞬时相互作用以促进它们的细胞分布。几种具有抑制能力的物质可以影响这种结合并对细胞造成损害。这个协议允许分析蛋白质是否与肌动蛋白丝或微管相互作用,以及在适用时,控制这种相互作用的条件。该测试基于目标蛋白和细胞骨架丝之间的特异性结合。如实施例图示(见下文)所示,测试从细胞裂解物开始,向其中加入肌动蛋白丝(由单体肌动蛋白产生)。然后孵育混合物(在由操作者选择的不同实验条件下进行),使得感兴趣的蛋白质(在实施例中,肌球蛋白)与肌动蛋白丝结合。然后将样品离心以从肌动蛋白丝分离未结合或弱结合的蛋白质,感兴趣的蛋白质和最终痕量较少特异性的蛋白质与其结合。

关键字:肌动蛋白丝, 微管, 肌球蛋白, 蛋白结合


材料和试剂

  1. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(≥97.0%)(Sigma-Aldrich,目录号:E3889)
  2. 鸟苷5'-三磷酸钠盐水合物(GTP)(≥95%)(Sigma-Aldrich,目录号:G8877)
  3. DL二硫苏糖醇(DTT)(≥99.0%)(Sigma-Aldrich,目录号:43819)
  4. 腺苷5'-三磷酸镁盐(ATP)(95-98%)(Sigma-Aldrich,目录号:A0770)
  5. PIPES二钠盐(≥99%)(Sigma-Aldrich,目录号:P3768)
  6. 甘油(≥99%)(Sigma-Aldrich,目录号:G5516)
  7. MgCl 2(≥98%)(无水)(Sigma-Aldrich,目录号:M8266)
  8. 蔗糖(≥99.5%)(Sigma-Aldrich,目录号:S9378)
  9. 紫杉醇(来自短叶红豆杉的紫杉醇)(≥95%)(Sigma-Aldrich,目录号:T7402)
  10. 牛微管蛋白(冻干,> 99%纯)(CYTOSKELETON,目录号:TL238-B)
  11. 兔骨骼肌的肌动蛋白蛋白(冻干,> 99%纯)(CYTOSKELETON,目录号:AKL99)
  12. 2D Quant Kit(通用电气公司)或Bradford蛋白质测定试剂盒(Bio-Rad Laboratories)
  13. 微管蛋白稀释缓冲液(TDB)(参见配方)
  14. 甘油缓冲液(GB)(参见配方)
  15. "蔗糖缓冲液"缓冲液(SC)(参见配方)
  16. 一般肌动蛋白缓冲液(A缓冲液)(参见配方)
  17. 聚合诱导剂(PI)(参见配方)
  18. F-肌动蛋白的缓冲缓冲液(参见配方)

设备

  1. 用于裂解细胞和组织的装置(用于细胞裂解的装置的类型取决于一个正在工作的细胞系统;动物细胞可以容易地裂解,而植物细胞由于细胞壁的存在而需要更多的能量方法 例如在液氮下冷冻和用杵和砂浆粉化,因此不可能给出精确的指示,但我们倾向于选择个体操作者。)
  2. 电泳装置(Mini-PROTEAN II电泳细胞)(Bio-Rad Laboratories)
  3. 装备有70Ti固定角转子和用于4ml聚合管的适配器的超速离心机(Beckman Coulter,型号:Optima LE-80K)
  4. Bio-Rad Mini-Trans-Blot细胞仪(可选)

软件

  1. ImageJ或Bio-Rad数量一

程序

  1. 微管结合测定
    1. 可溶(细胞质)提取物的制备
      1. 该步骤必须取决于其上正在工作的细胞或组织。理论上,在复杂组织的情况下,使用塑料Eppendorf杵或Ultra-Turrax匀浆器,可以在裂解缓冲液中直接裂解软组织(例如动物组织)。对于植物细胞,优选在液氮下冷冻感兴趣的组织,然后用研杵和研钵研磨。在组织完全粉末化后,使液氮蒸发。将组织粉末移至合适的管,如eppendorf管或15-ml锥形管(取决于起始材料的量)。加入裂解缓冲液,将样品在4℃或冰上温育15分钟,伴随温和搅拌。最后,将样品高速离心(> 100,000×g),用于沉淀细胞碎片并在上清液中获得胞质蛋白。虽然可溶性蛋白质的提取物可以在液氮下冷冻并储存在-80℃,我们总是喜欢使用新鲜材料,因为难以估计提取物的时间稳定性。裂解缓冲液的组成可以从物种调整到物种,请参考关于特定组织的文献,以确定最佳裂解缓冲液。
      2. 确定细胞质提取物的蛋白质浓度,以混合已知的蛋白质量。几种商业试剂盒可用于测定蛋白质浓度(例如2D Quant Kit或Bradford Protein Assay试剂盒)。总是取等分试样进行电泳。
    2. 微管的制备
      1. 解冻冻干的微管蛋白(动物或植物来源,1毫克)一等分,然后加入200微升TDB,使最终浓度为5毫克/毫升。
      2. 加入25μl的GB,并在35℃下孵育20分钟
      3. 将1.8ml TDB与81μl500μM紫杉醇(TDB-T)混合。 在35℃下孵育15-20分钟,使紫杉醇溶液的温度与微管蛋白相同。
      4. 在孵育结束时,向225μl的微管蛋白样品中加入1.8ml TDB-T。 轻轻混合。 在0.5mg/ml的浓度下,微管蛋白的最终体积为≈2ml。
      5. 通过将1.8ml TDB与81μl500μM紫杉醇混合制备另外的TDB-T
    3. 准备绑定组合。
      混合细胞溶质提取物(可变量,例如从0到40   微升,以便工作范围在10-100微克 蛋白)与微管样品的恒定体积(40μl) 微升)。 使用剩余卷添加其他 物质(即抑制剂),然后将体积调整为100   微升与TDB-T缓冲液。 体积可以相应地增加   以适应不同的转子和管
    4. 在室温下孵育样品30分钟。
      如果是实验中检查的变量,则可以调整试验温度。
    5. 细胞骨架细丝的分离
      1. 在20℃下将样品在100,000×g离心40分钟,超过0.5ml蔗糖缓冲缓冲液。
      2. 离心后,上清液位于蔗糖垫上方,因此可以用移液管除去而不接触蔗糖垫。 处理上清液进行电泳。
      3. 将颗粒直接重悬于适于电泳分析的变性缓冲液(例如Laemmli缓冲液)(最大100微升)。
    6. 分析
      通过电泳测试单个级分(上清液和沉淀) (SDS-PAGE)和可能通过蛋白质印迹,后者被要求 具体确定和量化感兴趣的蛋白质
  2. 肌动蛋白丝结合测定
    1. 细胞裂解液的制备
      1. 它取决于特定的组织或细胞,一个正在工作。 有关详细信息,请参阅先前的协议。
      2. 使用商业试剂盒(如2D Quant Kit或Bradford Protein Assay试剂盒)确定样品的蛋白质浓度,并始终收集一个等分试样进行电泳。
    2. 肌动蛋白丝的制备
      1. 解冻冻干肌动蛋白(动物或植物来源,1毫克)一等分
      2. 通过加入100μl的A缓冲液将肌动蛋白稀释至10mg/ml的浓度。从管中取出溶液(通常是Eppendorf管),并置于5ml或15ml管中
      3. 用A缓冲液(100μl肌动蛋白+ 2.4ml A-缓冲液)以0.4mg/ml的浓度稀释肌动蛋白。在冰上孵育1小时(不需要搅拌)。
      4. 加入聚合诱导剂1x终浓度:2.25ml + 0.25ml肌动蛋白聚合诱导剂10x。在室温下孵育1小时
    3. 准备绑定组合。
      混合细胞溶质提取物(可变量,例如从0到40  微升,以便工作范围在10-100微克 蛋白质)与恒定体积的肌动蛋白丝样品(50 微升)。使用剩余卷添加其他 物质(即抑制剂),然后将体积调整为100  微升与A-缓冲液/聚合诱导剂混合物(这是 相同缓冲液的肌动蛋白溶液)。 可以增加体积 相应地适应不同的转子和管
    4. 在室温下孵育样品30-60分钟。
      如果是实验中检查的变量,可以调整试验温度
    5. 细胞骨架细丝的分离
      1. 在20℃下将样品以150,000×g离心60-90分钟,在0.5ml F-肌动蛋白缓冲缓冲液中。
      2. 取上清液并处理以进行电泳。
      3. 将沉淀重悬在100μl适合于电泳分析的变性缓冲液(例如Laemmli缓冲液)中。
    6. 分析
      1. 通过电泳测试单个级分(上清液和沉淀) (SDS-PAGE)和可能通过蛋白质印迹,后者被要求 特异性识别和定量感兴趣的蛋白质(图1) 1)。
      2. 在这两种情况下,可以使用图像采集系统和特定软件(免费如ImageJ或如Bio-Rad Quantity One)来进行蛋白质印迹分析后的条带的相对定量。


        图1.具有肌动蛋白丝的结合分析的实例。具体地,将来自兔骨骼肌(箭头)的蛋白质肌球蛋白与丝状肌动蛋白(箭头)一起温育。 S和P分别表示上清液和沉淀。泳道1,分子量标准,其值由左侧的数字指示。泳道2,肌球蛋白(市售)。泳道3-4,肌球蛋白与肌动蛋白丝孵育后得到的上清液和沉淀。泳道5-6,在5'-腺苷酰-β,γ-亚氨基二磷酸(AMPPNP,ATP的不可水解的类似物)存在下肌球蛋白与肌动蛋白丝孵育后获得的上清液和沉淀。泳道7-8,上清液和沉淀 在ATP存在下用肌动蛋白丝孵育肌球蛋白后获得。 注意,肌球蛋白在ATP存在下弱结合,但在AMPPNP存在下非常强

食谱

  1. 微管蛋白稀释缓冲液(TDB)
    80mM Pipes,pH 6.8
    1 mM EGTA
    1mM MgCl 2
    2 mM GTP
  2. 甘油缓冲液(GB)
    80mM Pipes,pH 6.8
    1 mM EGTA
    1mM MgCl 2
    60%(w/v)甘油
  3. "蔗糖缓冲液"缓冲液(SC)
    80mM Pipes,pH 6.8
    1 mM EGTA
    1mM MgCl 2
    10%(w/v)蔗糖 20μM紫杉醇
  4. 一般肌动蛋白缓冲液(A缓冲液)
    5mM Tris-HCl pH8.0
    0.2mM CaCl 2·h/v 0.2 mM ATP
    0.5 mM DTT
  5. 10x聚合诱导剂(PI)
    500 mM KCl
    20mM MgCl 2/
    10 mM ATP
  6. F-肌动蛋白缓冲液
    5mM Tris-HCl pH8.0
    2mM MgCl 2/
    50 mM KCl
    10%(v/v)甘油

致谢

该方案改编自以前发表的文章:Del Duca等人(2013)。这项工作得到了意大利大学和研究部资助的PRIN 2007(授予号2007RZCW5S_003)和PRIN 2008(授予号2008BK7RXB)和博洛尼亚大学(RFO 2010 [授予号RFO10DELDU]和RFO 2011 [授权号RFO11DELDU])到SDD

参考文献

  1. Cai,G.,Faleri,C.,Del Casino,C.,Emons,A.M。和Cresti,M。(2011)。 烟草花粉管中胼lose质合酶,纤维素合酶和蔗糖合酶的分布受到不同方式的控制肌动蛋白丝和微管。 植物生理学 155(3):1169-1190。
  2. Del Duca,S.,Faleri,C.,Iorio,R.A.,Cresti,M.,Serafini-Fracassini,D.and Cai,G.(2013)。 梨花粉管中转谷氨酰胺酶在细胞骨架和膜动力学方面的分布。 植物生理学 161(4):1706-1721。
  3. Del Duca,S.,Serafini-Fracassini,D.,Bonner,P.,Cresti,M.and Cai,G。(2009)。 由花粉转谷氨酰胺酶催化的翻译后修饰对微管和肌动蛋白丝的功能性质的影响。/a> Biochem J 418(3):651-664。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Del Duca, S. and Cai, G. (2013). Binding Assay of Cytosolic Proteins to the Cytoskeleton. Bio-protocol 3(22): e980. DOI: 10.21769/BioProtoc.980.
  2. Del Duca, S., Faleri, C., Iorio, R. A., Cresti, M., Serafini-Fracassini, D. and Cai, G. (2013). Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics. Plant Physiol 161(4): 1706-1721. 
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