Format

Send to

Choose Destination
Curr Protoc Plant Biol. 2018 Sep;3(3):e20071. doi: 10.1002/cppb.20071. Epub 2018 Aug 14.

Profiling Interactome Networks with the HaloTag-NAPPA In Situ Protein Array.

Yazaki J1,2,3, Galli M1,2, Kim AY1,2, Ecker JR1,2,4.

Author information

1
Genomic Analysis Laboratory, Salk Institute for Biological Studies, La Jolla, California.
2
Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California.
3
RIKEN Center for Integrative Medical Sciences, Yokohama City, Japan.
4
Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California.

Abstract

Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones.

KEYWORDS:

HaloTag-NAPPA; cell-free protein expression; in situ protein array; interactome; protein-protein interaction

PMID:
30106517
DOI:
10.1002/cppb.20071

Supplemental Content

Loading ...
Support Center