Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics
采用滑动分析法分析基于微管的运动蛋白动力学
发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2210 浏览次数: 14430
评审: Jyotiska ChaudhuriTamara VellosilloJason A. Neidleman
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
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Abstract
The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.
Keywords: Gliding assay (滑动分析法)
Mitotic motor (有丝分裂马达蛋白)
in vitro motility (体外运动)
Polarity-marked microtubules (标记极性的微管)
Glass chamber (玻璃室)
Background
Mitotic spindles are protein machinery that dominate mitosis. The mitotic spindle utilizes microtubule-based motor proteins to organize itself, and exert forces to drive cell division. Microtubule-based motor proteins produce mechanical work using energy derived from ATP hydrolysis (Coppin et al., 1997). Motor proteins translocate microtubules in a unidirectional manner. The behavior of motility can be observed by in vitro gliding assay (Tao and Scholey, 2010), in which the motors are affixed onto a glass surface and supplied with microtubules and ATP. The motility of the motor proteins can then be studied using fluorescence microscopy and the details of their dynamic behavior can be observed in real time. This updated protocol will allow analysis of microtubule-based motor protein function with the use of in vitro microtubule gliding assays (Tao et al., 2006 and 2016).
Materials and Reagents
- Pipette tips (Corning, catalog number: 4860 )
- Centrifuge tubes (Corning, catalog number: 430290 )
- Thickwall polycarbonate tubes (Beckman Coulter, catalog number: 343775 )
- Coverslips (Sigma-Aldrich, catalog number: Z692263 )
- Microscope slides (Fisher Scientific, catalog number: S17466A )
- Coverslips (Fisher Scientific, catalog number: S175211A )
- Guanosine-5’-[(α,β)-methyleno]triphosphate (GMPCPP) (10 mM) (Jena Bioscience, catalog number: NU-405S )
- Tubulin (CYTOSKELETON, catalog number: TL238A )
- Rhodamine tubulin (CYTOSKELETON, catalog number: TL590M )
- Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
- N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876 )
- Guanosine 5’-triphosphate (GTP) (Sigma-Aldrich, catalog number: G8877 )
- Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: 295426 )
- Casein (Sigma-Aldrich, catalog number: C7078 )
- ATP (Sigma-Aldrich, catalog number: A6419 )
- Paclitaxel (Taxol) (Sigma-Aldrich, catalog number: T7402 )
- Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
- EGTA (Sigma-Aldrich, catalog number: E3889 )
- Tris base (Sigma-Aldrich, catalog number: T1503 )
- Potassium chloride (KCl) (Sigma-Aldrich: P5405 )
- Protease inhibitors:
Aprotinin (Sigma-Aldrich, catalog number: A3428 )
Benzamidine (Sigma-Aldrich, catalog number: B6505 or 12072 )
Note: The product “ B6505 ” has been discontinued.
Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Leupeptin (Sigma-Aldrich, catalog number: L2884 )
Soybean trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
Tert-Amyl methyl ether (TAME) (Sigma-Aldrich, catalog number: 283096 ) - Catalase (Sigma-Aldrich, catalog number: C1345 )
- Glucose oxidase (Sigma-Aldrich, catalog number: C6766 or G7141 )
Note: The product “ C6766 ” has been discontinued. - Glucose (Sigma-Aldrich, catalog number: D9434 )
- Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5368 )
- BRB80 buffer solution (see Recipes)
- Buffer L (see Recipes)
- Anti-fade solution (see Recipes)
Equipment
- Micropipette
2-20 μl (Sigma-Aldrich, catalog number: Z717304 )
10-100 μl (Sigma-Aldrich, catalog number: Z717312 ) - Ultra-centrifuge (Beckman Coulter, model: TLA-100 )
- NanoDrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
- Fluorescence microscope (Nikon Instruments, model: Eclipse E600 )
- Water bath (Thermo Fisher Scientific, model: PrecisionTM General Purpose Baths , catalog number: TSGP02)
Software
- ImageJ (Version 1.51J, https://imagej.nih.gov/ij/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Shim, A., Tezuka, G., Kupcha, L. and Tao, L. (2017). Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics. Bio-protocol 7(7): e2210. DOI: 10.21769/BioProtoc.2210.
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分类
细胞生物学 > 细胞成像 > 荧光
生物化学 > 蛋白质 > 荧光
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