Ambergen Technology

Figure 6: PC-PRINT of Proteins to Microarray Substrates.

(A.) High density PC-PRINT microarrays. NeutrAvidin or BSA coated 10 µm beads were used to capture a test protein that was dual labeled with PC-Biotin and Cy5 fluorescence. PC-PRINT was then performed. +Light = PC-PRINT with proper near-UV light treatment; -Light = PC-PRINT in the absence of the proper light treatment. (B.) PC-PRINT based protein-protein interaction assays. Human proteins were cell-free expressed and TRAMPE labeled with both PC-biotin and BODIPY-FL fluorescence (green). Proteins were captured on NeutrAvidin agarose beads (~100 µm) followed by PC-PRINT onto activated microarray substrates. Printed proteins: -DNA = samples differing only by omission of the DNA from the expression reaction; MDM = ubiquitin-protein ligase E3 MDM2; GST = glutathione-s-transferase. The microarray was probed with a p53-Cy5 conjugate ("p53 probe"; red fluorescence). In the "Mix" panel only, MDM and GST bait proteins were separately cell-free expressed, TRAMPE labeled with PC-biotin only and, prior to isolation, each mixed with crude expressed p53 probe that was TRAMPE labeled with BODIPY-FL only (green). Proteins and complexes were isolated onto beads via the PC-biotin, the beads pooled and then PC-PRINT was performed. This printed array was then probed with a Cy5 conjugated antibody (red) against a common epitope in all printed proteins. The "Mix" panel is a 2-color overlay.

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