Protein arrays

1. LEAPS technology Protein arrays An array of 110 different antibodies incubated with various levels of the fluorescently labelled cognate antigens in a serum background. (Courtesy Dr Brian Haab, The Van Andel Research Institute, Grand Rapids, MI, USA)

2. Introduction • Detect proteins • Monitor their expression levels • Investigate protein interactions and functions

3. Production of a Protein Array Coated surface Robotic protein application Scan Incubation with fluorescent labelled sample Arrayed proteins

4. Defining characteristics Protein arrays are solid-phase ligand binding assay systems using immobilised proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. highly parallel (multiplexed) and often miniaturised (microarrays, protein chips).

5. Advantages • Rapid • Automatable • Capable of high sensitivity • Economical on reagents • Giving an abundance of data for a single experiment.

6. Areas of application • 1.Diagnostics: detection of antigens and antibodies in blood samples; • profiling of sera to discover new disease markers; environment and • food monitoring. • 2.Proteomics: protein expression profiling; organ and disease specific • arrays. • 3.Isolation of individual members from display libraries for • further expression or manipulation: selection of antibodies and • protein scaffolds from phage or ribosome display libraries for use in • capture arrays. • 4.Protein functional analysis: protein-protein interactions; • ligand-binding properties of receptors; enzyme activities; • antibody cross reactivity and specificity, epitope mapping.

7. Protein sources • Cell-based expression systems for recombinant proteins • Purification from natural sources • Production in vitro by cell-free translation systems • Synthetic methods for peptides • Many of these methods can be automated for high throughput production. • For capture arrays and protein function analysis, it is important that proteins • should be correctly folded and functional; this is not always the case, e.g. • where recombinant proteins are extracted from bacteria under denaturing conditions. • Nevertheless, arrays of denatured proteins are useful in screening antibodies for • cross-reactivity, identifying autoantibodies and selecting ligand binding proteins.

8. Formats and surfaces Miniaturisation of familiar immunoassay methods such as ELISA and dot blotting. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

10. Protein immobilisation considerations • Variables in immobilisation of proteins include: • The coupling reagent • The nature of the surface being coupled to • The properties of a good protein array support surface are: Should be chemically stable before and after the coupling procedures, • Allow good spot morphology • Display minimal nonspecific binding • Not contribute a background in detection systems • Be compatible with different detection systems.

11. Protein immobilisation considerations The immobilisation method used should be: Reproducible Applicable to proteins of different properties (size, hydrophilic, hydrophobic) Amenable to high throughput and automation Compatible with retention of fully functional protein activity

18. Large-scale protein arrays Protein array containing 192 purified human proteins on glass. Incubation of the slide with anti-RGSHis antibody reveals the recombinant human proteins Detected with a Cy3-labeled secondary antibody.

19. Challenges and Bottlenecks