Journal
ANALYTICAL BIOCHEMISTRY
Volume 438, Issue 2, Pages 104-106Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.03.029
Keywords
Phos-tag; Antibody; Sandwich assay; Phosphorylation; Signal transduction; Cell lysate
Funding
- Japan Society for the Promotion of Science [22390006, 22590037, 24590050]
- Ministry of Education Culture, Sports, Science, and Technology [23117522]
- Grants-in-Aid for Scientific Research [24590050, 22590037, 22390006] Funding Source: KAKEN
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We describe two procedures for performing sandwich assays of phosphorylation of protein multiplexes by using several antibodies and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer. The first procedure is based on an antibody microarray technique with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. The second is based on the Bio-Plex suspension array technique with a flow-based microplate fluorescence reader system. By using this procedure, we demonstrated the quantitative detection of the entire level of phosphorylation in a target protein involved in intracellular signaling. (C) 2013 Elsevier Inc. All rights reserved.
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