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Optimization of Solid-Phase Synthesis of the 1-40 Beta-Amyloid and Preparation of Antibodies Revealing It under Immunoblotting Conditions

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Abstract

The solid-phase synthesis and purification of the 1-40 sequence of the human beta-amyloid were optimized, resulting in a preparation of a product with a high yield and homogeneity more than 95%. The synthetic peptide is capable of forming oligomers. This fact was confirmed by electrophoresis in the polyacrylamide gel with a subsequent immunoblotting and fluorescence spectrophotometry using the thioflavin T dye. An available method for a production of the highly specific anti-beta-amyloid antibodies with a high titer was developed. These antibodies recognized both monomeric and oligomeric forms of the 1-40 peptide of beta-amyloid under the immunoblotting conditions.

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Funding

This study was supported by the Russian Foundation for Basic Research, project no. 19-04-00624.

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Authors and Affiliations

  1. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia

    T. D. Volkova, D. O. Koroev, A. V. Kamynina, M. P. Filatova & O. M. Volpina

  2. Belozersky Research Institute of Physicochemical Biology, Moscow State University, 119234, Moscow, Russia

    A. V. Avetisyan

Authors
  1. T. D. Volkova
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  2. D. O. Koroev
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  3. A. V. Kamynina
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  4. M. P. Filatova
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  5. A. V. Avetisyan
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  6. O. M. Volpina
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Corresponding author

Correspondence to T. D. Volkova.

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COMPLIANCE WITH ETHICAL STANDARDS

This article does not contain any studies involving human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of Interests

The authors declare that they have no conflicts of interest.

Additional information

Translated by L. Onoprienko

Abbreviations: Аβ, beta-amyloid; Fmoc, 9-fluorenylmethoxycarbonyl; KLH, keyhole limpet hemocyanine; PBS, the phosphate buffered saline that contained 137 mM NaCl, 2.7 mM KCl, 8 mM NaH2PO4, and 1.5 mM KH2PO4 (рН 7.4); TBTU, tetrafluoroborate of О-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea; ThT, the thioflavin fluorescent dye; Trt, trityl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

Corresponding author: phone: +7 (495) 336-57-77; e-mail: tdvol@mx.ibch.ru.

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Volkova, T.D., Koroev, D.O., Kamynina, A.V. et al. Optimization of Solid-Phase Synthesis of the 1-40 Beta-Amyloid and Preparation of Antibodies Revealing It under Immunoblotting Conditions. Russ J Bioorg Chem 46, 217–222 (2020). https://doi.org/10.1134/S1068162020020181

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  • DOI: https://doi.org/10.1134/S1068162020020181

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