Work

A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions

Year: 2012

Type: article

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.

Source: Analytical Biochemistry

DOI: 10.1016/j.ab.2012.01.034

Authors: Yi Sun, Marcus Gallagher-Jones, Colin Barker, Gavin J. Wright

Institution: Wellcome Sanger Institute

Language: English

Topic: Advanced Biosensing Techniques and Applications

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,540

Funder: Wellcome Trust