Binding Assay of Cytosolic Proteins to the Cytoskeleton
胞质蛋白与细胞骨架相互作用研究
发布: 2013年11月20日第3卷第22期 DOI: 10.21769/BioProtoc.980 浏览次数: 13303
评审: Tie Liu
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
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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.
Microtubule (微管)
Myosin (肌球蛋白)
Protein binding (蛋白结合)
Materials and Reagents
- Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (≥ 97.0%) (Sigma-Aldrich, catalog number: E3889 )
- Guanosine 5’-triphosphate sodium salt hydrate (GTP) (≥ 95%) (Sigma-Aldrich, catalog number: G8877 )
- DL-Dithiothreitol (DTT) (≥ 99.0%) (Sigma-Aldrich, catalog number: 43819 )
- Adenosine 5’-triphosphate magnesium salt (ATP) (95-98%) (Sigma-Aldrich, catalog number: A0770 )
- PIPES disodium salt (≥ 99%) (Sigma-Aldrich, catalog number: P3768 )
- Glycerol (≥ 99%) (Sigma-Aldrich, catalog number: G5516 )
- MgCl2 (≥ 98%) (anhydrous) (Sigma-Aldrich, catalog number: M8266 )
- Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S9378 )
- Taxol (Paclitaxel from Taxus brevifolia) (≥ 95%) (Sigma-Aldrich, catalog number: T7402 )
- Bovine Tubulin (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: TL238-B )
- Actin protein from rabbit skeletal muscle (lyophilized, > 99% pure) (CYTOSKELETON, catalog number: AKL99 )
- 2D Quant Kit (General Electric Company) or Bradford Protein Assay kit (Bio-Rad Laboratories)
- Tubulin Dilution Buffer (TDB) (see Recipes)
- Glycerol buffer (GB) (see Recipes)
- “Sucrose cushion” buffer (SC) (see Recipes)
- General actin buffer (A-buffer) (see Recipes)
- Polymerization Inducer (PI) (see Recipes)
- Cushion buffer for F-actin (see Recipes)
Equipment
- 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.)
- Electrophoretic apparatus (Mini-PROTEAN® II Electrophoresis Cell) (Bio-Rad Laboratories)
- Ultracentrifuge (Beckman Coulter, model: Optima LE-80 K) equipped with a 70 Ti fixed-angle rotor and adapters for 4-ml polyallomer tubes
- Bio-Rad Mini-Trans-Blot Cell Apparatus (optional)
Software
- ImageJ or Bio-Rad Quantity One
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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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分类
植物科学 > 植物细胞生物学 > 细胞结构
生物化学 > 蛋白质 > 相互作用 > 蛋白质-蛋白质相互作用
植物科学 > 植物生物化学 > 蛋白质 > 相互作用
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