样品制备单病毒粒子的原子力显微镜和超分辨率荧光成像

1Department of Physics & Astronomy, University of Utah, 2Center for Cell and Genome, University of Utah
Published 1/02/2014
0 Comments
  CITE THIS  SHARE 
Biology
 

Summary

Cite this Article

Copy Citation

Hodges, J. A., Saffarian, S. Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging. J. Vis. Exp. (83), e51366, doi:10.3791/51366 (2014).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

病毒粒子对玻璃表面的固定化是在单个病毒粒子成像的关键步骤。在这里,我们提出了从单分子成像实验,允许单个病毒粒子粘附到玻璃表面具有特异性采用的技术。此制备是基于接枝的玻璃表面与PLL-G-PEG和PLL-G-PEG-生物素的混合物,增加了一层抗生物素蛋白,最后通过连接生物素化的病毒特异性抗体的产生病毒体的锚。我们已经将该技术应用在一系列的实验,包括原子力显微镜(AFM)和超分辨率荧光成像。此样品的制备方法导致的病毒粒子的表面的附着力控制。

Materials

Name Company Catalog Number Comments
Glass coverslips (fPALM) Electron Microscopy Services 72225-01 25 mm Coverslips
PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)  SuSoS - Stock: 0.5 mg/ml in PBS
NeutrAvidin biotin-binding protein Invitrogen A2666 Stock: 0.25 mg/ml in PBS
Alexa Fluor 647 goat anti-rabbit IgG (H+L)  Invitrogen A21245 1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer
α-VSV-G  Invitrogen ab34774 1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer
Gluox Sigma G2133-250KU Glucose oxidase type seven from Aspergillius 
Catalase Sigma C40-100mg Catalase from Bovine liver 
MEA Sigma 30070-10G Cysteamine (MEA
Stock Buffer 50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose
NTE 10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA
Slide-A-Lyzer, mini dialysis unit Thermo Scientific 10,000 MW
Microscope Cover Glass: 35 CIRCLE #1 Fisherbrand 35 CIRCLE #1

DOWNLOAD MATERIALS LIST

References

  1. Pang, H. -B., et al. Virion stiffness regulates immature HIV-1 entry. Retrovirology. 10, 4 (2013).
  2. Kol, N., et al. A Stiffness Switch in Human Immunodeficiency Virus. Biophys. J. 92, 1777-1783 (2007).
  3. Ortega-Esteban, A., et al. Minimizing tip–sample forces in jumping mode atomic force microscopy in liquid. Ultramicroscopy. 114, 56-61 (2012).
  4. Ortega-Esteban, A., et al. Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue. Sci. Rep. 3, (2013).
  5. Hernando-Pérez, M., et al. Physical Virology: Direct Measurement of Phage. phi29 Stiffness Provides Evidence of Internal Pressure (Small 15/2012). Small. 8, 2365-2365 (2012).
  6. Carrasco, C., et al. DNA-mediated anisotropic mechanical reinforcement of a virus. Proc. Natl. Acad. Sci. U.S.A. 103, 13706-13711 (2006).
  7. Sofia, S. J., Premnath, V. V., Merrill, E. W. Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption. Macromolecules. 31, 5059-5070 (1998).
  8. Visnapuu, M. -L., Duzdevich, D., Greene, E. C. The importance of surfaces in single-molecule bioscience. Mol. Biosyst. 4, 394-403 (2008).
  9. Elenko, M. P., Szostak, J. W., Van Oijen, A. M. Single-molecule binding experiments on long time scales. Rev. Sci. Instrum. 81, (2010).
  10. van Oijen, A. M., et al. Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder. Science. 301, 1235-1238 (2003).
  11. Böcking, T., Aguet, F., Harrison, S. C., Kirchhausen, T. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat. Struct. Mol. Biol. 18, 295-301 (2011).
  12. Cocucci, E., Aguet, F., Boulant, S., Kirchhausen, T. The First Five Seconds in the Life of a Clathrin-Coated Pit. Cell. 150, 495-507 (2012).
  13. Rust, M. J., Bates, M., Zhuang, X. W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy. 3, 793-795 (2006).
  14. Betzig, E., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313, 1642-1645 (2006).
  15. Hess, S. T., Girirajan, T. P. K., Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 91, 4258-4272 (2006).
  16. Juette, M. F., et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods. 5, 527-529 (2008).
  17. Xu, K., Zhong, G., Zhuang, X. Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons. Science. 339, 452-456 (2013).
  18. Huang, B., Wang, W., Bates, M., Zhuang, X. Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy. Science. 319, 810-813 (2008).
  19. Hodges, J., et al. Asymmetric packaging of polymerases within vesicular stomatitis virus. Biochemical and Biophysical Research Communications. 440, 271-276 (2013).
  20. Lawson, N. D., Stillman, E. A., Whitt, M. A., Rose, J. K. Recombinant vesicular stomatitis viruses from DNA. Proc. Natl. Acad. Sci. U.S.A. 92, 4477-4481 (1995).

Comments

0 Comments


    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Video Stats