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Nat Protoc. 2015 May;10(5):756-67. doi: 10.1038/nprot.2015.044. Epub 2015 Apr 16.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

Author information

1
The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.

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.

PMID:
25881200
PMCID:
PMC4464792
DOI:
10.1038/nprot.2015.044
[Indexed for MEDLINE]
Free PMC Article

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