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GrainGenes Reference Report: PYC-65-1777

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Reference	PYC-65-1777
Title	Identification of barley CK2 alpha targets by using the protein microarray technology
Journal	Phytochemistry
Year	2004
Volume	65
Pages	1777-1784
Author	Kramer A [ Show all 7 ]
Abstract	We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2 alpha . As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FASTTM slides. The generated protein microarrays were incubated with an expression library-derived barley CK2 alpha in the presence of ( gamma -33P)ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2 alpha as high mobility group proteins and calreticulin
External Databases	Pubmed: 15276436
Keyword	[ Hide all but 1 of 30 ] antibody assay barley casein kinase cdna cdna library ck2 alpha clone database filial tissues hmgb proteins homology hordeum vulgare kinase assay kinase ck2 maize microarray microarray technology phosphorylation phosphorylation of target proteins protein protein kinase protein microarray technology purification regulator sequence signal signal transduction substrate tissue

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