Figure 2: Protein microarray quality.

(a) Microarray with 2016 proteins that were expressed in situ; visualisation was by incubation with red and green labelled antibodies that recognise common N- or C-terminal epitopes, respectively. (b) The typical ratio is shown of foreground to background signals for the N- (left) and C-terminus (right); blue lines indicate regions of identical intensity. (c) Detection of the C-termini of the 2016 expressed proteins with Cy3-conjugated anti-V5 antibody; the horizontal line represents a signal of three standard deviations above background. (d) Determination of the amount of in situ synthesised GFP by comparison to spotted material of known concentration; twenty measurements each were done; the red line represents the average amount of synthesised protein plus/minus one standard deviation. (e) Microarrays of T. brucei proteins were incubated with the labelled, synthetic RNA sequences shown. The white circles highlight positive signals. On the lower array, also a second protein exhibited interaction. In (f), the interacting protein from the lower microarray was analysed in more detail in comparison to a derivative with one point mutation (Mut) and another, unrelated protein. The upper panel shows the protein amounts, detected by antibody binding to the N-terminus. The lower panel shows binding of the synthetic RNA. Subsequent studies demonstrated a more than 3,000-fold difference in affinity to the specific RNA between wildtype and mutated protein. (g) Binding to 74 human transcription factors of a labelled synthetic DNA sequence representing the TERT promoter (left). Mutation of one base pair in the binding sequence led to a very different binding pattern (right).