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. 2017 Jul 11;7:5070. doi: 10.1038/s41598-017-05336-7

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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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The 2B7/MW1 conformational immunoassay preferentially detects mHTT conformation in cocktails of isolated HTT proteins with wild type or mutant polyQ. (A) The 2B7/MW1 TR-FRET assay performed on wild type HTT, mHTT or a cocktail (of equivalent final protein concentration) of the two proteins at 20 °C (a) or measured after shifting the same samples to 4 °C (b). For simplicity, only results for a representative experiment performed on the N573 HTT protein fragments are shown. (B) Summary of data (ratio TR-FRET signals at 4 °C/20 °C) obtained on individual isolated HTT proteins (3 lengths, namely exon 1, N573 and full length) or cocktails of equivalent final protein concentration (Wt + Mut). (C) Same as A, obtained on the same proteins using a (control) TR-FRET immunoassay (2B7/4C9) which does not interrogate the polyQ region and does not detect a temperature- and polyQ dependent conformational change^{11, 12}, demonstrating efficient detection of all proteins. (D) Same as B (ratio TR-FRET signals at 4 °C/20 °C) obtained on the same proteins using a (control) TR-FRET immunoassay (2B7/4C9) which does not interrogate the polyQ region and does not detect a temperature- and polyQ dependent conformational change. In (B and D) values represent means and standard deviations of the means of three independent experiments (two-way ANOVA with Bonferroni’s post-test, degrees of significance are indicated).