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Native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen-ligand complexes
Claudia D'Ercole, Ario De Marco, 2024, objavljeni povzetek znanstvenega prispevka na konferenci

Opis: 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
Ključne besede: protein complexes, contact blotting, native agarose gels, protein interaction
Objavljeno v RUNG: 17.06.2024; Ogledov: 339; Prenosov: 0
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The fork remodeler helicase-like transcription factor in cancer development : all at once
Yossma Waheed, Aditya Mojumdar, Mohammad Shafiq, Ario De Marco, Matteo De March, 2024, pregledni znanstveni članek

Ključne besede: gene repair, cancer, genome stability
Objavljeno v RUNG: 12.06.2024; Ogledov: 372; Prenosov: 0
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Instruct-ERIC network : biophysical characterization of antigen-nanobody complexes
Claudia D'Ercole, 2024, objavljeni povzetek znanstvenega prispevka na konferenci

Opis: 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.
Ključne besede: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies
Objavljeno v RUNG: 31.05.2024; Ogledov: 535; Prenosov: 0
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Imagination is the limit for nanobody applications : lecture at the Zhejiang Lab for Regenerative Medicine, Vision and Brain Health, Zhejiang, March 21st 2024
Ario De Marco, 2024, predavanje na tuji univerzi

Ključne besede: nanobodies
Objavljeno v RUNG: 25.03.2024; Ogledov: 684; Prenosov: 0
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Implications of nanobody library design : lecture at the Zhejiang University, March 22nd 2024
Ario De Marco, 2024, predavanje na tuji univerzi

Ključne besede: nanobodies
Objavljeno v RUNG: 25.03.2024; Ogledov: 559; Prenosov: 0
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The long march of nanobodies : from freak elements to biotechnology stars
Ario De Marco, 2024, prispevek na konferenci brez natisa

Ključne besede: nanobodies
Objavljeno v RUNG: 25.03.2024; Ogledov: 592; Prenosov: 0
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