Protein microarray printing and 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).