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Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring th...

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Autores principales: Murphy, Patrick J. M., Shannon, Morgan, Goertz, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196170/
https://www.ncbi.nlm.nih.gov/pubmed/21788938
http://dx.doi.org/10.3791/3061
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author Murphy, Patrick J. M.
Shannon, Morgan
Goertz, John
author_facet Murphy, Patrick J. M.
Shannon, Morgan
Goertz, John
author_sort Murphy, Patrick J. M.
collection PubMed
description Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) (1,2). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation (3),such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase (4) and a repair enzyme (5), bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes (1,2) and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.
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spelling pubmed-31961702011-10-24 Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy Murphy, Patrick J. M. Shannon, Morgan Goertz, John J Vis Exp Bioengineering Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) (1,2). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation (3),such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase (4) and a repair enzyme (5), bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes (1,2) and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources. MyJove Corporation 2011-07-18 /pmc/articles/PMC3196170/ /pubmed/21788938 http://dx.doi.org/10.3791/3061 Text en Copyright © 2011, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Bioengineering
Murphy, Patrick J. M.
Shannon, Morgan
Goertz, John
Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title_full Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title_fullStr Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title_full_unstemmed Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title_short Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
title_sort visualization of recombinant dna and protein complexes using atomic force microscopy
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196170/
https://www.ncbi.nlm.nih.gov/pubmed/21788938
http://dx.doi.org/10.3791/3061
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