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. 2019 May 3;10:18. doi: 10.1186/s13100-019-0160-9

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 2 — Purification of RelSt3 protein and determination of its oligomeric state. a Different steps of RelSt3 purification from E. coli (12% SDS-PAGE). Crude extract (lane 1) was submitted to a His-Trap affinity chromatography. Flow-through analysis (lane 2) indicated that most of the protein was retained. The pool of imidazole elution fractions (lane 3) was then digested by the TEV protease to remove His-tag (lane 4), and the sample was then loaded on a second His affinity chromatography. Without tag, the protein was not retained (lane 5), and was concentrated to be injected on a gel filtration column. Finally, the resulting RelSt3-containing fractions were concentrated (lane 6). Lane 7 corresponds to the Spectra Multicolor Broad Range protein ladder (Thermo Scientific). The gel was stained with Coomassie Brilliant Blue. b Gel filtration (Sephadex 200) analysis of RelSt3 protein. The elution profile recorded showed a large main peak with a maximum elution volume of 72.4 ml. c Glutaraldehyde cross-linking of RelSt3 purified protein. 5 μg of RelSt3 protein were incubated without (lane 1) or with 0.001% (lane 2), 0.01% (lane 3), 0.025% (lane 4), 0.05% (lane 5) or 0.1% (lane 6) of glutaraldehyde. The products were then analyzed by SDS-PAGE (10%), with a Coomassie Brilliant Blue staining. See Materials and Methods for details. Lane 7: Spectra Multicolor Broad Range protein ladder. d Analysis of the dispersion state of RelSt3 by analytical ultracentrifugation. Sedimentation velocity of RelSt3 was analyzed at 5 μM. The diagram represents the sedimentation coefficient distribution [c(s)]