Chapter title |
DIGE-ABPP by Click Chemistry: Pairwise Comparison of Serine Hydrolase Activities from the Apoplast of Infected Plants.
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Chapter number | 15 |
Book title |
Plant-Pathogen Interactions
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Published in |
Methods in molecular biology, March 2014
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DOI | 10.1007/978-1-62703-986-4_15 |
Pubmed ID | |
Book ISBNs |
978-1-62703-985-7, 978-1-62703-986-4
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Authors |
Hong TN, van der Hoorn RA, Tram Ngoc Hong, Renier A. L. van der Hoorn, Hong, Tram Ngoc, Hoorn, Renier A. L. van der |
Abstract |
Activity-based protein profiling (ABPP) is a targeted functional proteomics method that displays the active proteome by using small molecule probes that react covalently with the active sites of protein classes. Comparison of activity profiles from two different samples is not always easy, especially when using probes that generate too many signals. For accurate comparison of protein activities between two proteomes, we developed difference gel electrophoresis ABPP (DIGE-ABPP), which compares two fluorescently labeled proteomes in the same gel lane. This protocol describes the labeling of two proteomes with alkyne-labeled probes, followed by the coupling with two different fluorophores using "click chemistry," the separation of mixed proteomes on protein gels, and the quantification and comparison of the activity profiles. We applied DIGE-ABPP to investigate differential serine hydrolases activities in the apoplast of Nicotiana benthamiana challenged with Pseudomonas syringae p.v. tomato DC3000. |
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