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)

Introduction

Detect proteins

Monitor their expression levels

Investigate protein interactions and functions

Production of a Protein Array

Coated surface Robotic proteinapplication

Arrayedproteins

Incubation withfluorescent

labelled sample

Scan

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).

Rapid

Automatable

Capable of high sensitivity

Economical on reagents

Giving an abundance of data for a single experiment.

Advantages

Areas of application1. 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.

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.

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.

Company Architectures

GyrosCD centrifugation devices based on developments

in microfluidics

Biotrove microchannels in a plate

Zyomyx 3D posts on a silicon surface

Bio-Rad colour coding for microbeads

Quantum Dots semiconductor nanocrystals

Smartbeads barcoding for beads

Nanoplex Technologies

multimetal microrods

BioArray Solutions

beads assembled into planar arrays on semiconductor chips

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 bindingNot contribute a background in detection systems Be compatible with different detection systems.

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

Protein immobilisation considerations

Company DetectionFrom DNA array different fluorophores (e.g. Cy-3, Cy-5)

PerkinElmer Lifesciences

tyramide signal amplification (TSA)

Zeptosens planar waveguide technology

Bio-Rad phycoerythrin as label [Luminex]

Molecular Staging rolling circle DNA amplification

HTS Biosystems, Intrinsic Bioprobes

surface plasmon resonance

Ciphergen, Intrinsic Bioprobes

mass spectrometry

Genicon Sciences

BioForce Laboratories

resonance light scattering

atomic force microscopy

Large-scale protein arrays

Incubation of the slide with anti-RGSHis antibody reveals the recombinant human proteins

Detected with a Cy3-labeled secondary antibody.

Protein array containing 192 purified human proteins on glass.

Challenges and Bottlenecks