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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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Glutaraldehyde efficiently fixes insulin on the blotted membrane. (a,b) Reference standard of insulin (1 µg) was subjected to Tris/Tricine/urea SDS-PAGE. After electro-blotting, the one blotted membrane was separated into 1 slip for the control experiment (Coomassie brilliant blue [CBB] staining immediately after electro-blotting, labeled as “Immediately after blotting”) and 5 slips for the aldehyde treatment. Paraformaldehyde (PFA) (a) or glutaraldehyde (GA) (b) concentrations were verified as shown. N.T. indicates a control treatment. (c) Reference standards of proinsulin (200 ng) and insulin (125 ng) were subjected to Tris/Tricine/urea SDS-PAGE. After electro-blotting, the blotted membrane was separated into 3 slips to verify the effect of the retrieval step on insulin Western blotting (WB). Upper panels are the results of WB of insulin, and the lower panels are the results of CBB staining of the blotted membrane. To compare the effects of various conditions, WB images in each sub-part of the figure were captured at one time.