Assay Set-up

The assay is based on three sequential steps, i.e. the incubation of the sample with the affinity protein, the quantitative separation of free and protein-bound compounds, and the dissociation of the protein-ligand complexes. Ligands released from the protein are subsequently detected by LC-MS. 

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We have demonstrated the feasibility of miniaturized MS assays by converting the cathepsin B assay described in Section 5.2.2 to a chip format, using the same substrate and products for the MS-based readout [27]. The assay set-up is identical to the format described in Fig. 5.1. The advantages of chips as micro reactors over fused silica capillaries are in their compactness, strength, greater degrees of freedom in design and material, and the presence of hair-pin curves to increase the diffusion rate. 

For each multiplex, prepare a mixture of the required primers. The final concentration of each primer in the primer mix should be 9 xM. Consider how much primer mix you will need so that this step has to be performed only once for the assay set-up. Each single reaction (i.e., a single well in a 384-well microplate) requires 1 pL of primer mix. 

Several of the assay principles can also be used in liquid assays making such assay set-ups possible where clones after propagation in small cultures can be screened in pools. Such systems can be automated for high throughput screening systems. 

As measurements of interactions often happen in dissected systems where molecules are tested in other than the original context, it should be crucial to test a model in conceptually different assay set-ups to validate findings and try to eliminate experimental artefacts. These requirements can be difficult to achieve for the carbohydrate-carbohydrate interaction even more difficult than to find the natural components of protein-carbohydrate interaction which is already a difficult task as demonstrated by the hunt for the real selectin ligands [103]. Much of older literature on carbohydrate-carbohydrate interaction does not necessarily hold the requirements for unambiguous testing in different systems, but due to lack of possible alternatives at the time they were performed they have been included in this summary. Improvement of methods and technology should give the chance to test these concepts in future. 

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