Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Substrate loading experiments

SG Sebastian Groß
BS Bastien Schnell
PH Patrick A. Haack
DA David Auerbach
RM Rolf Müller
request Request a Protocol
ask Ask a question
Favorite

All substrate loading experiments were performed in 50 µL scale using 25 mM TRIS (pH 7.5), 150 mM NaCl, 10 mM MgCl2 and 1 mM ATP. For CysH, CysH∆AMDH, CysK-M1-4, and CysG-M5-6, 5 µM protein and 1 mM amino acid were used to test loading of different substrates. Hydroxylation of CysH-bound l-asparagine by CysJ was tested by additionally supplementing 1 mM α-KG, 50 μM FeSO4, and 500 nM CysJ. To analyze the transfer of l-asparagine from CysH to CysB, 1 μM CysH was incubated with 1 mM l-asparagine and 5 μM CysB. The transfer of l-asparagine from CysB to CysK-M3 was tested by incubating 5 µM CysK-M3, 1 μM CysH, 500 nM CysB with 1 mM L-asparagine. All reactions were incubated for either 5 min or 2 h at RT, respectively. Direct intact protein UPLC-ESI-MS analysis was done as described below.

Copyright and License information: The Author(s) ©2021
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