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. 2017 Jul 31;7:6949. doi: 10.1038/s41598-017-04456-4

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© The Author(s) 2017

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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During the general WB method, insulin is easily washed out from the blotted membrane compared with proinsulin. The indicated doses of proinsulin and insulin were separated by Tris/Tricine/urea SDS-PAGE. (a) WB was performed by using anti-insulin clone c27c9, which recognizes both proinsulin and insulin (upper panel). After WB detection, the membrane was stained with CBB R-250 (lower panel). In the condition, insulin was below detection limit of CBB staining of the membrane. (b) The PVDF membrane immediately after electro-blotting for WB (upper panel) was stained with CBB R-250. As another experiment, the separated gel was directly stained with CBB R-250 (lower panel).