Development of Arabidopsis Protein Chip

Plant Microarray Printing & Probing

For printing, purified recombinant protein preparations were re-arrayed and printed from 384-well plates. Each well contained at least 10 µl of each protein preparation at a concentration of approximately 1 µg/µl. Each protein was arrayed in quadruplicate spots onto glass slides coated with nitrocellulose polymer (FAST slides; Schleicher & Schuell) with a 48-pin contact printer (Bio-Rad Chip Writer Pro). The arrayed proteins were air dried over-night at 4ºC and transferred to -80ºC for storage. Control spots were printed with vector-only preparations and wild type tissue purified as described. All printed slides were used for biochemical assays within 2-3 weeks of the printing date. Slides were blocked in SuperBlock Blocking Buffer (Pierce) for 1-2 hours at room temperature and washed in TBS-1% Tween (TBS-T) for 3 times, 10 min each wash. Blocked slides were incubated with primary monoclonal anti-cMyc IgG (Santa Cruz) diluted 1:2,500 in SuperBlock Buffer solution for 1 hour at room temperature, washed 3 times 10 min each in TBS-T, and incubated in the secondary antibody, an Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratoires, Inc.), for 1 hour at room temperature. After the final wash, 3 times 10 min in TBS-T, the slides were dried by spinning at 1,000 x g for 1 min, and scanned in a Genepix 4200A slide scanner (Axon Instruments).