Simultaneous measurement of 10,000 protein-ligand affinity constants using microarray-based kinetic constant assays

James P. Landry, Yiyan Fei, Xiangdong Zhu

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Fluorescence-based endpoint detection of microarrays with 10, 000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10, 000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (k on, k off, andk a=k on/k off) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this binding curve assay platform is comparable to those at the National Institutes of Health Molecular Library Screening Centers, making it a practical method in screening compound libraries for novel ligands and for system-wide affinity profiling of proteins, viruses, or whole cells against diverse molecular targets.

Original languageEnglish (US)
Pages (from-to)250-259
Number of pages10
JournalAssay and Drug Development Technologies
Volume10
Issue number3
DOIs
StatePublished - Jun 1 2012

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Ligands
Fluorescence
Endpoint Determination
Proteins
National Institutes of Health (U.S.)
Libraries
Viruses
Technology

ASJC Scopus subject areas

  • Drug Discovery
  • Molecular Medicine

Cite this

Simultaneous measurement of 10,000 protein-ligand affinity constants using microarray-based kinetic constant assays. / Landry, James P.; Fei, Yiyan; Zhu, Xiangdong.

In: Assay and Drug Development Technologies, Vol. 10, No. 3, 01.06.2012, p. 250-259.

Research output: Contribution to journalArticle

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