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. 2018 Jul 20;8:10990. doi: 10.1038/s41598-018-29253-5

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© The Author(s) 2018

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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In vitro conjugation of DARPin.X-TEFCA and PDZ1. (A) PDZ1 (100 µM) was incubated with different DARPin.X-TEFCA constructs or the negative control DARPin.9.26-TEFSA (100 μM) at room temperature for 4 h. The mixture was then separated on 12% SDS-PAGE gels under reducing or non-reducing conditions. The conjugation products (indicated by black arrows) can only be seen under non-reducing condition (lane 3, 7). (B) The percentage of PDZ1 reacted with different DARPin.X.TEFCA was calculated by dividing the intensity of unreacted PDZ1 band in lane 3 or 7 with that in lane 1. Values and error bars represent the average and standard deviation, respectively, of at least two independent experiments.