PUBLICATION
A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions
- Authors
- Sun, Y., Gallagher-Jones, M., Barker, C., Wright, G.J.
- ID
- ZDB-PUB-240502-24
- Date
- 2012
- Source
- Analytical biochemistry 424: 455345-53 (Journal)
- Registered Authors
- Wright, Gavin J.
- Keywords
- none
- MeSH Terms
-
- Humans
- Protein Interaction Mapping/economics
- Protein Interaction Mapping/methods*
- Extracellular Space/metabolism*
- Recombinant Proteins/chemistry
- Recombinant Proteins/isolation & purification
- Recombinant Proteins/metabolism*
- Zebrafish
- Protein Array Analysis/economics
- Protein Array Analysis/methods*
- Molecular Sequence Data
- Cost-Benefit Analysis
- Immobilized Proteins
- Tissue Culture Techniques
- Protein Binding
- HEK293 Cells
- Biotinylation
- Animals
- Zebrafish Proteins/metabolism
- Amino Acid Sequence
- Streptavidin/metabolism
- PubMed
- 22342946 Full text @ Anal. Biochem.
Citation
Sun, Y., Gallagher-Jones, M., Barker, C., Wright, G.J. (2012) A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions. Analytical biochemistry. 424:455345-53.
Abstract
Low-affinity extracellular protein interactions are critical for cellular recognition processes, but existing methods to detect them are limited in scale, making genome-wide interaction screens technically challenging. To address this, we report here the miniaturization of the AVEXIS (avidity-based extracellular interaction screen) assay by using protein microarray technology. To achieve this, we have developed protein tags and sample preparation methods that enable the parallel purification of hundreds of recombinant proteins expressed in mammalian cells. We benchmarked the protein microarray-based assay against a set of known quantified receptor-ligand pairs and show that it is sensitive enough to detect even very weak interactions that are typical of this class of interactions. The increase in scale enables interaction screening against a dilution series of immobilized proteins on the microarray enabling the observation of saturation binding behaviors to show interaction specificity and also the estimation of interaction affinities directly from the primary screen. These methodological improvements now permit screening for novel extracellular receptor-ligand interactions on a genome-wide scale.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping