1. Native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen-ligand complexesClaudia D'Ercole, Ario De Marco, 2024, published scientific conference contribution abstract Abstract: Background. Protein complexes provide valuable biological information but can be difficult to handle [1]. Therefore, technical advancements designed to improve their manipulation are al-ways useful.
Methods. We investigated the opportunity to exploit native agarose gels and the contact bot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes [2]. As a simple model of protein-protein interactions, an anti-gen-ligand complex was used in which both proteins were fused to reporters.
Results. At each step, it was possible to visualize both the antigen, fused to a fluorescent pro-tein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers.
Conclusions. The procedure is rapid, inexpensive, and the several detection opportunities al-lows for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific train-ing.
References:
[1] O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Séraphin. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. (San Diego, Calif.), 24(3), 218–229
[2] C. Sakuma, M. Nakagawa, Y. Tomioka, T. Maruyama, K. Entzminger, J.K. Fleming, T. Shibata, Y. Kurosawa, C.J. Okumura, T. Arakawa, T. Akuta. Western blotting of native proteins from agarose gels. Biotechniques. 2022 May;72(5):207-218
Keywords: protein complexes, contact blotting, native agarose gels, protein interaction
Published in RUNG: 17.06.2024; Views: 348; Downloads: 0
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2. Instruct-ERIC network : biophysical characterization of antigen-nanobody complexesClaudia D'Ercole, 2024, published scientific conference contribution abstract Abstract: Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature which may lead to their massive decline [1]. Understanding the molecular mechanisms of specific stress conditions and monitoring the fluctuations of reliable forest plant biomarkers with affordable methods would be instrumental for assessing stress levels over the time. Ascorbate peroxidase (APX) represents a suitable plant biomarker. APX is a hydrogen peroxide-scavenging enzyme the critical role of which has been described in several plants, both herbaceous and woody. Its activity generally increases under oxidative stress during which its peroxide detoxifying function is part of the wider ascorbate-glutathione cycle [2]. The development of reagents to detect such fluctuations would help the evaluation of plant physiological conditions. In this study, nanobodies (Nbs) targeting APX have been identified. Nbs correspond to the variable domain of heavy chain-only antibodies derived from camelids. They are small (15 kDa), stable, and can be easily produced in bacteria fused to different protein tags according to the downstream applications [3]. After their isolation by biopanning against soluble APX, they have been produced and underwent a biophysical characterization in combination with their antigen (APX-Nb complex) to identify the best binders in terms of stability and affinity. The protein complex characterization was supported by Instruct-ERIC and mainly performed at the BIOCEV institute of Prague. Data from Mass Photometry and Dynamic Light scattering evidenced the formation of the protein complexes, whereas the preliminary data of Hydrogen-Deuterium Exchange Mass Spectrometry, performed with the aim of identifying the residues involved in the paratope/epitope interface, were insufficient to clarify the issue and rather suggested that the interaction has low affinity. This indication was then confirmed by ELISA assay. The combination of multiple methods allowed a comprehensive sample characterization which will require further structural analyses to provide a complete picture of the APX-Nb complex.
[1] G. Marie. B. C. M. B. C. Walters, “Forest decline and tree mortality in a southeastern Ohio oak-hickory forest,” Ohio Journal of Science , vol. 97, 1997.
[2] O. Chew, J. Whelan, and A. H. Millar, “Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants,” Journal of Biological Chemistry, vol. 278, no. 47, 2003, doi: 10.1074/jbc.M307525200.
[3] S. Muyldermans, “A guide to: generation and design of nanobodies,” FEBS J, vol. 288, no. 7, pp. 2084–2102, Apr. 2021, doi: 10.1111/febs.15515.
Keywords: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies
Published in RUNG: 31.05.2024; Views: 545; Downloads: 0
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3. A practical guide for the quality evaluation of fluobodies/chromobodiesUrša Štrancar, Claudia D'Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March, Ario De Marco, 2024, original scientific article Keywords: fluorescent proteins, protein stability, protein degradation
Published in RUNG: 16.05.2024; Views: 453; Downloads: 2
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4. Biological applications of synthetic binders isolated from a conceptually new adhiron libraryClaudia D'Ercole, Matteo De March, Gianluca Veggiani, Sandra Oloketuyi, Rossella Svigelj, Ario De Marco, 2023, original scientific article Keywords: synthetic library, adhirons, affimers, C-reactive protein, SpyCatcher
Published in RUNG: 17.10.2023; Views: 1101; Downloads: 3
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7. Macroporous epoxy-based monoliths functionalized with anti-CD63 nanobodies for effective isolation of extracellular vesicles in urineJulia Neumair, Claudia D'Ercole, Matteo De March, Martin Elsner, Michael Seidel, Ario De Marco, 2023, original scientific article Keywords: extracellular vesicles, monolith chromatography, nanobodies, functionalitazion strategies
Published in RUNG: 27.03.2023; Views: 1477; Downloads: 11
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8. Real-time monitoring of Arundo donax response to saline stress through the application of in vivo sensing technologyJanni Michela, Claudia Cocozza, Federico Brilli, Sara Pignattelli, Filippo Vurro, Nicola Coppede, Manuele Bettelli, Davide Calestani, Francesco Loreto, Andrea Zappettini, 2021, original scientific article Abstract: One of the main impacts of climate change on agriculture production is the dramatic increase of saline (Na+) content in substrate, that will impair crop performance and productivity. Here we demonstrate how the application of smart technologies such as an in vivo sensor, termed bioristor, allows to continuously monitor in real-time the dynamic changes of ion concentration in the sap of Arundo donax L. (common name giant reed or giant cane), when exposed to a progressive salinity stress. Data collected in vivo by bioristor sensors inserted at two different heights into A. donax stems enabled us to detect the early phases of stress response upon increasing salinity. Indeed, the continuous time-series of data recorded by the bioristor returned a specific signal which correlated with Na+ content in leaves of Na-stressed plants, opening a new perspective for its application as a tool for in vivo plant phenotyping and selection of genotypes more suitable for the exploitation of saline soils.
Keywords: Arundo donax, saline stress, vivo sensing technology
Published in RUNG: 17.12.2021; Views: 1974; Downloads: 18
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9. "Umetnost je zgodovina napak, ki pa so poetične, ki delujejo kot nekaj izvirnega. Za vsemi napakmi se skrivajo zgodbe." : "UMETNOST JE ZGODOVINA NAPAK, KI PA SO POETIČNE, KI DELUJEJO KOT NEKAJ IZVIRNEGA. ZA VSEMI NAPAKMI SE SKRIVAJO ZGODBE."2021, radio or television broadcast, podcast, interview, press conference Abstract: Med gosti letošnjega festivala Fabula – Literature sveta je leta 1984 rojena italijanska pisateljica Claudia Durastanti. V slovenščino imamo preveden njen roman Tujka, avtofikcijsko pripoved, ki se začne z odraščanjem ob gluhih starših; odvija se od New Yorka, prek revne južne Italije do Rima in Londona – hkrati pa gre za razmisleke o identiteti, umetnosti in življenju.
Keywords: italijanske pisateljice, romani, intervjuji
Published in RUNG: 05.11.2021; Views: 1834; Downloads: 7
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