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Improving biomaterials imaging for nanotechnology : rapid methods for protein localization at ultrastructural level
Cano-Garrido, Olivia (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Garcia-Fruitos, Elena (Institut de Recerca i Tecnologia Agroalimentàries. Departament de Producció de Remugants)
Villaverde Corrales, Antonio (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Sánchez Chardi, Alejandro (Universitat Autònoma de Barcelona. Servei de Microscòpia)

Data: 2018
Resum: The preparation of biological samples for electron microscopy is material- and time-consuming because it is often based on long protocols that also may produce artifacts. Protein labeling for transmission electron microscopy (TEM) is such an example, taking several days. However, for protein-based nanotechnology, high resolution imaging techniques are unique and crucial tools for studying the spatial distribution of these molecules, either alone or as components of biomaterials. In this paper, we tested two new short methods of immunolocalization for TEM, and compared them with a standard protocol in qualitative and quantitative approaches by using four protein-based nanoparticles. We reported a significant increase of labeling per area of nanoparticle in both new methodologies (H = 19. 811; p < 0. 001) with all the model antigens tested: GFP (H = 22. 115; p < 0. 001), MMP-2 (H = 19. 579; p < 0. 001), MMP-9 (H = 7. 567; p < 0. 023), and IFN-γ (H = 62. 110; p < 0. 001). We also found that the most suitable protocol for labeling depends on the nanoparticle's tendency to aggregate. Moreover, the shorter methods reduce artifacts, time (by 30%), residues, and reagents hindering, losing, or altering antigens, and obtaining a significant increase of protein localization (of about 200%). Overall, this study makes a step forward in the development of optimized protocols for the nanoscale localization of peptides and proteins within new biomaterials.
Ajuts: Ministerio de Economía y Competitividad RTA2012-00028-C02-02
Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-132
Nota: Altres ajuts: AV thanks the granting of an ICREA ACADEMIA award. EGF thanks CERCA Programme (Generalitat de Catalunya), and European Social Fund for supporting our research. OCG received a PhD fellowship from MECD (FPU) and EGF a post-doctoral fellowship from INIA (DOC-INIA). The authors also acknowledge Micalis Institute INRA, France that kindly provides us the strain clpP-hrtA- NZ9000 (patent n°EP1141337B1/US6994997B1).
Drets: Tots els drets reservats.
Llengua: Anglès
Document: Article ; recerca ; Versió acceptada per publicar
Publicat a: Biotechnology Journal, Vol. 13, issue 4 (April 2018) , art. 1700388, ISSN 1860-7314

DOI: 10.1002/biot.201700388

21 p, 363.5 KB

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Documents de recerca > Documents dels grups de recerca de la UAB > Centres i grups de recerca (producció científica) > Ciències de la salut i biociències > Institut de Biotecnologia i de Biomedicina (IBB)
Articles > Articles de recerca
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 Registre creat el 2021-02-12, darrera modificació el 2022-03-15

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