Format

Send to

Choose Destination
Anal Biochem. 2012 May 1;424(1):45-53. doi: 10.1016/j.ab.2012.01.034. Epub 2012 Feb 17.

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

Author information

1
Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

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.

PMID:
22342946
PMCID:
PMC3325482
DOI:
10.1016/j.ab.2012.01.034
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center