הכנת דוגמאות לבודדה Virion כוח האטומי מיקרוסקופית ורזולוצית הסופר דימות פלואורסצנטי

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

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