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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

HS He Sun *
YL Yuanyuan Luo *
YM Yansong Miao

 (*contributed equally to this work) 发布: 2018年12月05日第8卷第23期 DOI: 10.21769/BioProtoc.3102 浏览次数: 6679

评审: Andrea PuharSam-Geun KongAnonymous reviewer(s)

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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) search Pyrene Assay (芘荧光检测) search Actin Polymerization (肌动蛋白聚合) search Actin Nucleation (肌动蛋白成核化) search Formin (甲酸精) search

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

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

生物化学 > 蛋白质 > 分离和纯化

生物化学 > 蛋白质 > 自组装

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