Microarrays—Solutions to the Protein Challenge

Abstract

The study of the proteome presents many challenges to the researcher. One of these is finding the best way to study tens of thousands of proteins in as short a time as possible. With the advances in miniaturization and automation over the recent past, several approaches are now available for producing high-density protein arrays on custom slides. After the arraying process is complete, lessons learned in the automated hybridization and imaging of cDNA and oligonucleotides now allow the consistent high-throughput study of antibody/antigen interactions.

Overview

Many scientists are making the transition from genomics to proteomics with extremely promising results. It has taken many years to develop products of the sophistication required to address the miniaturization and automation needs of the genomics market. As such, current microarray technology allows for simultaneous investigation of thousands of parameters within a single experiment. Scanning and analysis systems, based on fluorescence, chemiluminescence, mass spectrometry, radioactivity, or electrochemistry, are then used to detect if a biomolecular complex has been formed. The development of protein microarray technology now offers an important advance for the miniaturization and automation of protein/protein interaction studies.

Challenges to the Development of Protein Arrays

There are four major challenges to the development of protein arrays. These are:

1. Antibodies are the most commonly used capture mole-cules, but producing monoclonal antibodies in large amounts is both labor-intensive and expensive.

2. Proteins have a tendency to adsorb non-specifically to solid substrates. This can cause background problems (less sensitivity and low signal-to-noise ratio).

3. Proteins are complex, and maintaining their native state and orientation during immobilization needs to be optimized such that their ability to interact is maintained. Also, the buffers, salt, temperature, and humidity conditions required for optimal activity must be considered.

4. Beyond the arraying process, researchers are also keen to find ways to automate the resulting large number of antibody/antigen binding studies directly on the slide surfaces and then visualize those slides in high-resolution laser-scanning systems.

Finding systems that can respond to all these requirements has been difficult, but over the past ten years Genomic Solutions® has developed a suite of products that can readily address the arraying, binding, and visualization needs of the proteomics market.

Solutions Through Automation

Antibody Production

These challenges have been approached in several ways. For example, the HiGro® from Genomic Solutions is an incubation system specifically designed to grow cultures in microtiter plates (1). The ability to reduce culture volumes, coupled with a small orbit and controlled temperature and gas flow, results in fast cell growth and high levels of protein expression (2).

Protein Adsorption

Several slide chemistries are available to solve the issues of binding and orientation. These include membrane-based technologies from Schleicher and Schuell (FAST® and CAST™), and proprietary surfaces such as Genomic Solutions OmniGrid® Epoxy and Aldehyde slides, Accelr8′s OptArray™-Protein slides, NUNC™′s polymer coated glass slides, and the PerkinElmer™ HydroGel™ slides. It is also possible to use the new generation of compartmentalized slides such as the Nexterion™ slides from Schott, or microtiter plates from companies such as Corning and The Gel Company.

Protein Arraying

The currently accepted method for printing protein arrays is to use a non-contact system for spot deposition. To address this requirement, Genomic Solutions manufacture their proprietary SynQUAD™ solenoid valve technology (3). This technology has been in use for protein spot deposition for several years (4). Although piezo-dispensing technologies can print at higher densities, concerns remain over the heat production from the piezo crystal (bad for delicate proteins) or blockages from viscous products. This latter issue requires highly expensive vision correction systems. SynQUAD shows robustness, reproducibility, and speed.

Figure 1. The Genomic Solutions arrayers include Soft Touch Technology, Cooled Plate Storage, and Sonicating Wash Baths.

One myth regarding protein microarraying is the assumption that it is not possible to use contact printers to manufacture quality protein microarrays. This is incorrect, as shown by the large and growing number of publications and commercial products that are being produced using contact printers. Indeed, the first experiments carried out in the field of protein microarrays were from contact printing instruments, such as the Flexsys (now known as the GeneMachines® G³ Multi-Functional Workstation) from Genomic Solutions (5,6). Now, most of the world′s leading microarray laboratories are carrying out their research using contact printing systems. Even considering the more viscous solutions needed, both solid or split pins can be used as long as specific printing parameters, such as touch-off speed, can be controlled. In addition, the incorporation of sonicating water baths does assist in the cleaning process of the pins.

An important factor to consider when setting out to array proteins comes from the choice of slide chemistry and spotting solution; combining this with environmental considerations has a major effect on array quality. The ability to cool the arrayer and control the local humidity is extremely significant in the production of high-quality protein microarrays. Using the plate chiller in the BioBank area of the MicroGrid II system from Genomic Solutions acts to reduce the internal temperature of the instrument, and enhances successful protein arraying.

Three of the more popular slide types for protein microarray production are the Genomic Solutions OmniGrid Epoxy Slides, the FAST system from Schleicher and Schuell, and the HydroGel systems from PerkinElmer. It has been said that it is not possible to use these surface types with contact printers for microarray production. This too is not actually the case, and for use in protein microarrays, both the HydroGel and nitrocellulose (7) substrates are compatible with all Genomic Solutions microarray printing technology. The contact arrayers from the OmniGrid and MicroGrid ranges allow the researcher to control the spotting speed (using Soft Touch with user selectable speeds) when printing on such delicate surfaces to prevent damage. Adjustable target height then allows for the differences in the thickness of the many slide and microtiter plate types now available.

Figure 2. A selection of solid and quill pins are available for all arraying products.

Binding and Imaging

Once the arrays have been produced, there is still the need to study antibody/antigen interactions. The Genomic Solutions HybStation is a programmable automated hybridization and wash station typically used for cDNA and oligonucleotide microarray hybridizations. Its versatility also allows it to be used for carrying out antibody/antigen interactions (5). If fluorescent tags have been used, then the results can be imaged in the two-laser (red/green) or four-laser (red/green/blue/yellow) UC4 scanners with a 1-µm resolution and the ability to read four slides at a time (5).

Figure 3. The large numbers of antibody/antigen binding studies generated need to be visualized and analyzed.

In conclusion, there are several challenges to the production of high-quality proteins and their subsequent arraying, binding, and imaging. Genomic Solutions offers a suite of high-throughput, automated tools together with applications support and advise based on over ten year s experience to help the researcher meet the challenges associated with studying the proteome.

For more information or help and advice on designing your protein arrays, please send your requests to:

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