Figure 1. Detection methods for abundance-based protein microarrays. (a) Capture microarrays can be analyzed by chemically modifying the sample with fluorescent (or other readable) markers before applying the sample to the microarray. This has the advantage that all features of the microarray can be assessed simultaneously, but the limitation that any cross-reactivity in the analyte specific reagents (ASRs) used to capture the analytes will give false readings (blue feature). (b) Alternatively, the analytes can be detected by capture with one ASR and detection with a second specific to a different epitope in sandwich immunoassay fashion. This significantly reduces false readouts (blue feature), but can be more cumbersome to multiplex. (c) In the reverse-phase protein blot, the complex experimental sample itself is printed and probed with an ASR. This allows the rapid screening of many samples, but is highly subject to the specificity of the detecting ASR.

Figure 2. Assembly methods used to produce function-based protein microarrays. (a) Expressed and purified proteins can be affixed directly to the surface of a chemically activated matrix. By this method, native protein can be used and the proteins will tend to position in random orientations, such that on average, each surface is likely to be exposed to the interacting sample. However, the close attachment to the surface may limit the overall solvent exposure of the protein and the chemical linkage may affect protein folding. Fusion peptide tags added at the N- or C-terminus affix the protein through an affinity capture reagent. Proteins are produced either by (b) separate expression and purification or (c) by simultaneous expression and capture of the protein on the array surface. The use of fusion tags allows the protein to be held at a distance from the matrix, exposing more overall surface area to solvent, but sterically blocking either the N- or C-terminus and requiring the addition of fusion tags to all target proteins.