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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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Treatment of blocking buffer in pre-fixed blots enhances the improvement of WB signals from insulin. (a,b) Dilution series (panel a for low doses: 10, 3, 1, 0.3 ng and panel b for high doses: 100, 30, 10, 3 ng) of insulin were subjected to Tris/Tricine/urea SDS-PAGE. M indicates molecular weight markers. For concentration evaluation, 4 slips from the one blotted membrane were treated with the indicated concentration of blocking solution. (c) For time optimization, the dilution series (10, 3, 1, 0.3, 0.1 ng) of insulin were subjected to Tris/Tricine/urea SDS-PAGE. Three slips from the one blotted membrane were treated with the 1x blocking solution for the indicated time. M indicates a lane for molecular weight markers. One of the containing proteins cross-reacts with anti-insulin antibodies. (d) Flow diagram of the improved proinsulin/insulin WB. To compare the effects of the various conditions, WB images in each sub-part of the figure were captured at one time.