Abstract:
:AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Yu X,LaBaer Jdoi
10.1038/nprot.2015.044subject
Has Abstractpub_date
2015-05-01 00:00:00pages
756-67issue
5eissn
1754-2189issn
1750-2799pii
nprot.2015.044journal_volume
10pub_type
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