Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Preparative BMOE cross-linking

SH Sandro Holzer
JY Jiangyu Yan
MK Mairi L. Kilkenny
SB Stephen D. Bell
LP Luca Pellegrini
request Request a Protocol
ask Ask a question
Favorite

The individual and double PriS E34C and PriX K110C mutants of Chimera were prepared as described above and adjusted to a protein concentration of 20 μM. BMOE to a final concentration of 40 μM was then added to the protein sample and incubated for 30 min at 25 °C before the absolute BMOE concentration in the reaction was increased to 80 μM and incubated for further 30 min. Subsequently the reaction was stopped by adding a final concentration of 5 mM DTT to the mixture followed by a 10 min incubation at 25 °C. If necessary, the protein was concentrated with a spin concentrator to a volume of 1–2 mL and subjected to a Superdex S200 16/600 size-exclusion chromatography column (GE Healthcare). The purified cross-linked protein was then used for gel-based activity assay as described above.

Copyright and License information: 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/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A