Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 9, Issue 2, Pages 112-121Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057103260592
Keywords
microarrayed compound screening (mu ARCS); high-throughput screening; AMP-activated protein kinase (AMPK); streptavidin affinity membrane (SAM (TM))
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A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (muARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM(TM)). For detection, the SAM(TM) was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibitors of AMPK.
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