Multiplex analysis of Src family kinase signaling by microbead suspension arrays

Mel Campbell, Wen Rong Lie, Jing Zhao, David Hayes, Jehangir Mistry, Hsing Jien Kung, Paul A. Luciw, Imran H. Khan

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

There is renewed interest in the Src family of protein tyrosine kinases (SFKs) as a result of their potential utility as molecular targets for cancer therapy. This protein family consists of 9 nonreceptor tyrosine kinases that, although implicated in a diverse array of cellular functions, possess a similar modular structure. Here we describe a simple and efficient multiplex microbead immunoassay (MMIA), based on Luminex® xMAP technology, which allows for the simultaneous detection of 8 phosphorylated SFKs in a single assay. Microbead sets identifiable by unique fluorescence were individually coated with antibodies specific for an individual SFK member. Detection of phosphorylated SFKs was accomplished using a secondary antibody directed against phosphotyrosine. The assay requires ≤ 10 μg of cell lysate or nanogram amounts of purified SFK. The use of a generic secondary antibody allows for the expansion of the assay to include any other tyrosine kinase for which a specific antibody exists. Using either mammalian cell lines or purified, recombinant kinases as the SFK source, we demonstrate the utility of the assay by evaluating the phosphorylation status of SFK members following several in vitro manipulations designed to modulate the phosphotyrosine content of the kinases. These results show that the SFK multiplex assay is a robust tool to investigate the function of SFKs in basic and potentially in clinical research.

Original languageEnglish
Pages (from-to)488-496
Number of pages9
JournalAssay and Drug Development Technologies
Volume8
Issue number4
DOIs
Publication statusPublished - Aug 1 2010
Externally publishedYes

Fingerprint

src-Family Kinases
Microspheres
Suspensions
Phosphotyrosine
Antibodies
Protein-Tyrosine Kinases
Phosphotransferases

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

Cite this

Multiplex analysis of Src family kinase signaling by microbead suspension arrays. / Campbell, Mel; Lie, Wen Rong; Zhao, Jing; Hayes, David; Mistry, Jehangir; Kung, Hsing Jien; Luciw, Paul A.; Khan, Imran H.

In: Assay and Drug Development Technologies, Vol. 8, No. 4, 01.08.2010, p. 488-496.

Research output: Contribution to journalArticle

Campbell, M, Lie, WR, Zhao, J, Hayes, D, Mistry, J, Kung, HJ, Luciw, PA & Khan, IH 2010, 'Multiplex analysis of Src family kinase signaling by microbead suspension arrays', Assay and Drug Development Technologies, vol. 8, no. 4, pp. 488-496. https://doi.org/10.1089/adt.2009.0255
Campbell, Mel ; Lie, Wen Rong ; Zhao, Jing ; Hayes, David ; Mistry, Jehangir ; Kung, Hsing Jien ; Luciw, Paul A. ; Khan, Imran H. / Multiplex analysis of Src family kinase signaling by microbead suspension arrays. In: Assay and Drug Development Technologies. 2010 ; Vol. 8, No. 4. pp. 488-496.
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