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. 2019 Nov 7;9:16272. doi: 10.1038/s41598-019-52655-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The generated recombinant TSPs and α₂δ-1_S variants show high degree of purity and integrity in Coomassie staining and western blot analyses. (A,D left) Representative Coomassie-stained gels and (B,C and D right) immunoblots of three full-length TSP proteins, all carrying an N-terminal double strep II-tag: TSP-2, TSP-4, and COMP (A,B); α₂δ-1_S variants carrying either an N-terminal (α₂δ-1_S NTST) or a C-terminal (α₂δ-1_S CTST) double strep II-tag (C); α₂δ-1_S NTST and α₂ peptide chain carrying an N-terminal double strep II-tag, α₂ NTST (D). Proteins were separated under non-reducing (−DTT) or reducing conditions (+DTT) on 4–15% (B), 10% (C), and 7% (D) polyacrylamide gels, respectively, while in (A) proteins were separated on 0.5% agarose (w/v)/3% polyacrylamide (w/v) composite gels without prior DTT treatment. Proteins were either stained with colloidal Coomassie stain (A,D left) or detected with the following primary antibodies after blotting: mouse anti-strep II-tag (B,C) or rabbit anti-α₂δ-1 (D right). Secondary antibodies included the polyclonal rabbit anti-mouse IgG (B,C) and swine anti-rabbit IgG (D right), both conjugated with horseradish peroxidase (see Supplementary Table S2 for further information). In all gels the molecular weight standard (in kDa) indicated on the left was PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) except for (A) in which both thyroglobulin (Sigma) and recombinant laminin-111 (kind gift from Prof. Dr. Monique Aumailley, Institute for Biochemistry II, Centre for Biochemistry, Medical Faculty, University of Cologne) were used.