A protein array consists of multiple proteins immobilized on a solid support. Miniaturized assays are performed against these proteins to inform interactions with ligands such as other proteins, antibodies, lectins, glycans, aptamers, lipids, DNA, RNA, small molecules, and peptides. To better understand how protein arrays vary in their design, and to learn about the different applications they support, we spoke with Dr. Mike Mao and Dr. David Fang from RayBiotech, a provider of array products. This article focuses on the utility and advantages of recombinant protein arrays.
Protein-based arrays are categorized according to the nature of the immobilized protein. Antibody arrays are used to capture target antigens from body fluids, culture medium, or tissue extracts and are typically used to detect multiple proteins (tens to hundreds) within a single sample. Recombinant protein arrays contain a far greater density of immobilized biomolecules, allowing for investigation of hundreds or thousands of interactions simultaneously. Lectin arrays have a similar capacity to antibody arrays and use bound lectins as capture agents to detect glycoproteins. Reverse phase protein arrays (RPPA) rely on fixed tissue lysates or fluids and are useful for biomarker discovery.
Image 1: Protein Arrays
Image 2: Antibody Arrays
While antibody arrays have the singular purpose of detecting protein concentrations, recombinant protein arrays can be adapted to a variety of high-throughput applications. For example, they are frequently used as high-throughput kinase assays to characterize drug specificity. Protein arrays also have utility for autoantibody profiling to characterize autoimmune diseases and certain cancers and are employed as serological assays to measure immune responses in allergy testing. Further applications include assessment of antibody selectivity during development, identification of infection, vaccine development, and evaluation of post-translational modifications. According to Fang, recombinant protein arrays are becoming more widely used, particularly for proteomic level research and discovery. To deliver this huge breadth of data, just 50–100 ng protein is typically immobilized per spot.
Although antibody arrays and recombinant protein arrays often use similar sample volumes, the higher density of recombinant protein arrays significantly reduces overall sample usage. “For researchers using our antibody arrays, we usually recommend a minimum of 50 µL sample for a typical 40-plex cytokine antibody array with a 2x dilution,” notes Mao. “For the detection of greater numbers of protein targets, multiple 40-plex arrays can be run in parallel to detect 1000 or more antigens. In comparison, for recombinant protein arrays just two 500-plex products are needed to generate a similar quantity of data.”
Using established techniques such as mass spectrometry for protein identification, highly abundant proteins often mask the detection of low-abundance targets. Contrastingly, recombinant protein microarrays are highly sensitive to targets at a wide range of concentrations. Since many informative proteins such as enzymes or transcription factors are often low-abundance, recombinant protein arrays can yield greater insight from precious sample material.
The maximum number of quantifiable targets that can be achieved in a single sandwich-based multiplex immunoassay is around 100, an unavoidable limitation attributable to antibody cross-reactivity. Mao explains that a reason for this is that detection antibodies are provided in solution and applied to the array as a mixture. “To avoid high background and the generation of misleading results, it is important to manage the number of readouts from an antibody microarray,” he says. “However, because no antibody cocktail is used in a protein array, cross-reactivity is less of an issue. Thus, the spot density can be much higher.”
In producing an antibody array, it is necessary to optimize each antibody individually to account for target affinity. Recombinant protein arrays can instead be built using equal amounts of each protein. “Because the immobilized proteins are printed at a far greater concentration than their presence in biological samples, they are already over-saturated,” explains Mao. “The practice of immobilizing recombinant proteins in excess greatly reduces the length of time needed for optimization.”
Protein synthesis and subsequent immobilization is typically quicker and more straightforward than performing a similar process with antibodies. “Gene cloning, protein purification, and protein array production are generally much simpler processes than antibody production,” says Fang. “This affords streamlined development of custom arrays to meet specific research requirements.”
With their simple, high-content format, protein arrays are a cost-effective solution to rapidly generate vast quantities of robust and reproducible data. With the capacity to synthesize high-quality recombinant proteins for immobilization, RayBiotech can support almost any field of scientific research. As protein arrays continue to open up new possibilities for discovery, this important technology is poised for further expansion.