Identification of barley CK2alpha targets by using the protein microarray technology.
We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. 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 FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha 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 CK2alpha as high mobility group proteins and calreticulin.
Department of Lehrach, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany.
Armin
A
Kramer
Tanja
T
Feilner
Alexandra
A
Possling
Volodymyr
V
Radchuk
Winfriede
W
Weschke
Lukas
L
Bürkle
Birgit
B
Kersten
D047390:Casein Kinase II / Q000032:analysis*; D015723:Gene Library; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D007118:Immunoassay; D008970:Molecular Weight; D040081:Protein Array Analysis / Q000379:methods*
D047390:Casein Kinase II