Table of Contents > Genomics > Protein microarrays Print

Protein microarrays


Related terms
Future research
Author information

Related Terms
  • Antibodies, antigens, aptamers, atomic force microscopy, cancer, cell lysates, chemiluminescence, colorimetry, combinatorial therapy, fluorescent detection, individualized therapy, ink-jetting, laser capture microdissection, mass spectrometry, microarray, molecular diagnostics, molecular profiling, peptides, phage lysates, photolithography, piezoelectric spotting, proteomics, receptors, recombinant proteins, robotic contact printing, rolling circle DNA amplification, sector array, surface plasmon resonance, thermal spotting.

  • Protein microarrays, or protein chips, are devices with different substances (proteins or DNA sequences) affixed to a solid substrate in a regular pattern which can bind target molecules for the purpose of rapid, parallel biochemical and genetic analysis.
  • Each spot on a microarray is designed to capture a different molecule. A sample of different proteins or DNA sequences is analyzed by running it across the array, allowing each spot to bind its respective target, and then tagging the bound molecules with other molecules which emit some sort of detectable signal (often light). The signal intensity of each spot is proportional to the quantity of bound target molecules. This allows for the detection and measurement (qualitative and quantitative analysis) of proteins from the sample of interest. The spot pattern image is then captured, analyzed, and correlated with biological information with the help of a computer.
  • Protein microarray technology has widespread applications, such as the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of protein kinases (an enzyme that alters or modifies other proteins). Phospholipids are compounds of fats (lipids) and phosphates. Microarrays have also been used for clinical diagnostics (e.g. cancer markers in blood), monitoring disease states (e.g. heart enzymes in blood), and assessing the effectiveness of drugs and other therapies.
  • The theoretical underpinning of the protein microarray originated with the development of microspot ligand-binding and antibody assays (immunoassay) in the late 1980s. Since the introduction of microarrays, the technology has rapidly advanced, expanding into genetic analysis with DNA microarrays. DNA microarrays are useful in a number of applications including quantifying gene expression and the identification of mutations. A ligand is an ion, a molecule, or a molecular group that binds to another specific chemical entity to form a larger complex under controlled conditions. Immunoassay is a laboratory analysis technique to detect and quantify a substance using the principle of antigen and antibody reaction. An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen and are known as antibodies or immune bodies. Gene expression is the process of converting genetic information in a sequence of DNA into a functional molecular product, such as protein or RNA (ribonucleic acid), i.e. the translation and transcription process.
  • Protein chips: The commonly used solid or physical support material for the microarray chips include: glass slides, silicon, microwells/nanowells (extremely small wells), nitrocellulose membranes, PVDF (poluvinylidene fluoride) membranes, and magnetic and other microbeads. These support materials are synthetic and specialized materials. A nitrocellulose membrane is a thin layer formed by a mixture of pulpy or cotton-like polymers (large chain of molecules) of cellulose, nitric acid, and sulfuric acids. PVDF is a specialty plastic material made from polymer of vinylidene fluoride (a chemical compound), which is highly non-reactive to chemicals and has good tensile strength
  • It is important that the chip surface have certain properties, including: the ability to immobilize the capture protein, maintain the conformation (structural arrangement) and the functionality of the protein, remain chemically stable before and after the coupling procedures (joining of capture proteins on physical support surface), good spot morphology (form and structure of the spot), achieve maximum binding capacity to the capture protein, display minimal nonspecific binding, and be compatible with different standard microarray equipment and detection systems.
  • Coupling (connecting or linking) reagents are important for proper immobilization of capture proteins, as orientation of the surface-bound protein is an important factor in successful binding and analysis. Some of the surface coatings used are amines, aldehydes, nitrocellulose, gel pads, and poly-L-lysine. Many of these coatings also have certain special properties, such as an affinity to specific capture proteins (affinity coatings). Some examples include silanes, streptavidin, and biotin.
  • Capture proteins: These protein binding spots form the basis of microarray chips for protein profile studies. The capture proteins may be uniform or of similar or different types within each spot. Such proteins may be antibodies, antigens, nucleic acids, drugs, recombinant proteins, peptides, aptamers, enzymes (biological catalysts), receptors, or cell or phage (virus) lysates. Recombinant proteins are derived from synthetic forms of DNA (recombinant DNA) made by transplanting genes from one species into the cells of a host organism of a different species. Peptides are the building blocks of proteins. Aptamers are DNA/RNA or peptide molecules that bind to specific proteins or metabolites (substances produced by a chemical reaction in the body). Receptors are molecular structures or sites on the surface or interior of a cell that binds with substances, such as hormones, antigens, or drugs. Lysates are the contents of a cell or virus released after it has been lysed (cut open) by certain chemicals.
  • An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response takes the form of proteins that are specific to an antigen and are known as antibodies or immune bodies. Monoclonal antibodies are produced by a single type of immune cells that recognize only one specific antigen. Polyclonal antibodies are a mixture of different antibodies derived from different cell types, but which act against a single specific antigen by recognizing different epitopes (binding sites). Both monoclonal and polyclonal antibodies are used based on the requirements of the particular microarray.
  • Detection methods (readout): A number of different detection methods are available to localize the reactive proteins on microarray chips. These methods include labeling (attaching) the protein or small molecular probes with fluorescent, affinity, or radioisotope tags detected by fluorescent, colorimetric, and chemiluminescent methods. The chemiluminescence method uses a chemical dye that binds with a protein (e.g. biotin) attached to the probe, which can be made to emit light via a chemical reaction which is then detected by luminescent imager. Similarly in the colorimetric detection method, a staining reagent is detected by a colorimeter.
  • Generally, fluorescent detection is the preferred method, as it is safe, effective, sensitive, and compatible with a number of commercially available CCD (charged-coupled device) cameras or microarray laser scanners. CCD cameras take fluorescent images by capturing light signals and translating them to electronic signals.
  • Affinity tags and photochemical tags can also be used to label microarray probes. Affinity tags are short sequences of amino acids that have been tagged with agents that have an attraction towards specific proteins, which makes them easier to be detected by colorimetry.
  • In certain protein interactions, the labels used for detection may interfere with the probe's ability to interact with the target protein. This can be overcome by the use of label-free detection methods, such as surface plasmon resonance (SPR), mass spectrometry, rolling circle DNA amplification, and atomic force microscopy. These detection methods are accurate and efficient, and are equivalent to detection methods with labels.
  • Surface plasmon resonance biosensors are devices that use an optical method to measure the refractive index and resonance of the protein chip surface. The refractive index is a measure of the bending (refraction) of a beam of light when it passes from one medium to another medium (such as from air to glass). The protein interaction spots on the chip exhibit a characteristic refractive index and resonance depending on the relative level of binding that has occurred. The values measured by an SPR biosensor can therefore be used to determine the pattern of protein capture across the microarray surface.
  • Mass spectrometry is an analytical technique used to identify molecules based on their mass. It is an important tool in proteomics (the study of proteins and their characteristics).
  • Rolling circle DNA amplification (RCA) is a technique generally used for the detection of protein antigens. In RCA, a circular piece of DNA is attached to an antibody and then amplified (mass produced). Multiple copies of circular DNA remain attached to the antibody, which can be detected.
  • Atomic force microscopy (AFM) is a technique which employs a very high resolution scanning probe microscope. AFM can be used to determine the surface structure of biological molecules.

  • Primarily, there are three types of protein microarrays which are used to study the biochemical activities of proteins: analytical microarrays, functional microarrays, and reverse phase microarrays.
  • Analytical protein microarray: The basic purpose of this type of protein microarray is to detect and measure (qualitative and quantitative profiling) the relative mixture of proteins present within a cell or tissue. These chips are used especially to monitor levels of protein expression (how much protein has been translated from a particular gene), characterize different forms of protein, and aid in the detection and identification of clinical diseases and disorders. For example, certain proteins that are suggestive of certain cancers can be detected by this method.
  • This type of array uses a library of antibodies, antigens, DNA or RNA aptamers, and affibodies (artificially synthesized proteins with a high binding capacity) as capture agents, arrayed on the glass slides, microwells, or microbeads. Aptamers are DNA/RNA or proteins that bind to a specific target molecule. The contents of a cell or tissue, serum (clear liquid of the blood), or other bodily fluid sample (e.g. urine), is applied to the chip for analysis. The capture agents selectively bind to the target proteins from the sample. These complexes of capture agents and target proteins are then detected with fluorescently labeled antibodies.
  • Antibody microarrays are the most common form of analytical microarray. Examples of applications include profiling responses to environmental stress and characterizing healthy versus disease tissues. Sandwich immunoassays are the method of choice for detecting low abundance proteins, such as cytokines, and protein modifications, as they have a high specificity and sensitivity. Cytokines are proteins that play a vital role in the immune response of the body.
  • Reverse phase protein microarray(RPA): In this type of array, cells are isolated and separated from various tissues of interest, and then broken down (lysed) to release the contents of the cells (lysate) by chemical processes. The lysate is spotted or arrayed onto the slide surface (e.g. nitrocellulose) with the help of a contact pin arrayer. Contact pin arrayers are very tiny robotic pins that deliver sub-nanoliter capture protein sample volumes directly onto the surface of microchips. The slide surface should have the ability to immobilize the capture protein and maintain its conformation (structural arrangement), and the functionality of the protein should be chemically stable before and after the coupling procedures (joining of capture proteins on the physical support surface). The slides are then searched and probed with a variety of antibodies against the target protein of interest. These probe antibodies are labeled with fluorescence for detection.
  • RPA requires half the number of antibodies to detect a protein of interest in comparison to analytical protein arrays, and has the advantage of requiring only a very small cell or tissue sample for analysis, thereby making it a more accurate and cost-effective process.
  • Applications of RPA include detecting the presence of altered proteins as a result of a disease condition. It can further be used to help identify which protein pathway may be dysfunctional or altered in a cell, so that a specific therapy can be recognized to target the altered protein pathway and treat the disease of interest.
  • Functional protein array: This type of array is used to study the biochemical activities of proteins as well as various protein interactions, such as protein-protein, protein-DNA, protein-RNA, protein-lipid (fat), protein-drug, protein-enzyme, protein-small molecules, and antigen-antibody interactions.
  • In this type of array, the individual spots of chips or arrays are composed of purified proteins. The protein chip is allowed to react with probes that are marked with fluorescent dye for easy detection, and then washed. Stable interactions are identified by scanning the slides for fluorescent spots, which are indicative of protein interactions.
  • Protein chip construction: Protein microarray fabrication may be an automated or manual process, involving the imprinting of externally synthesized and purified capture molecules on a biologically reactive film on an array or a slide surface. Alternatively, the capture molecules can be synthesized in place and directly imprinted onto the array. The spacing of the protein spots on the slide surface or microwells depends on the size of capture molecules (the larger the molecules the larger the spacing required). As an example, antibody arrays typically have 375 mm (micrometer) spacing. The automated fabrication process is computerized and is much faster, efficient, and cost-effective in than the manual process.
  • Printing extremely small quantities of capture molecules on a slide surface is challenging, as it must be ensured that good spot structural characteristics, required densities, and good biological attachment are reproducibly maintained across the array. This process has been simplified with the help of automated capture molecule imprinting methods, which include robotic contact printing, ink-jetting, piezoelectric spotting, and photolithography.
  • Robotic contact printing arrayers have very tiny pins that deliver sub-nanoliter capture protein sample volumes directly onto the surface of microchips. Limitations of contact printing robots include an inability to align their pins to prefabricated structures and a need to touch the array surface. Ink-jetting invloves non-contact robotic array printers that deposit nanoliters to picoliters of protein droplets to polyacralymaide gel packets and microwells. Polyacrylamide is a large compound that is formed from several small molecules (polymers) of the chemical acrylamide. Ink-jet microarrays or spotters are commonly of two types: piezoelectric and thermal. The piezoelectric spotting method involves delivery of the sample droplets on electrical stimulation, while thermal spotting involves sample delivery upon heating. In photolithography, ultraviolet (UV) light is passed through a transparent or translucent membrane, which acts as a stencil onto the surface of the array slide. The antibody or other capture molecules are imprinted only on the points on the array slide that are activated by the stenciled image.

  • Protein arrays are currently being employed in clinical trials in the field of drug development for the detection of protein biomarkers, which may help researchers and doctors tailor therapy, and characterize or predict the course of a disease. For example, multiple sclerosis is a chronic (long-term) disorder in which a person's immune system attacks the body's own nerve cells. Interferon beta (IFN-beta) is a drug used in the treatment of multiple sclerosis. According to a recent research study, a biomarker known as B-cell activating factor (BAFF), was used to evaluate the therapeutic response to IFN-beta in multiple sclerosis patients by utilizing the microarray analysis method.
  • Protein chip technology is also currently used by researchers to discover previously unknown functional proteins and to identify new functionalities for already well-characterized proteins. For example, prominin-1 is a protein that has been earlier identified for its role in the process of the formation of several cancers, such as cancer of the eye and nerve cells. A recent research study utilizing protein microarray analysis found that prominin-1 may also be used as a marker to distinguish cells that have the potential to become cancerous from normal cell populations.

  • General: The drive behind the rapid development of protein microarray technology is in large part related to its wide utility in the discovery of the basic functions and characteristics of proteins, as well as applications in clinical diagnostics (detection and identification of disease), therapeutics (treatment of disease), forensics (scientific investigation of criminal activities), food testing, and environmental analysis.
  • Protein biomarkers/tumor markers: Protein microarray technology has been used to discover novel protein biomarkers, which may help in the detection and/or diagnosis of disease. It may also aid in monitoring the course of a disease. Some examples of useful biomarkers include proteins associated with certain cancers, such as of the breast, intestines, or skin. Tumor markers can be analyzed and quantified from very minimal biopsy material (extracts from suspected cancerous tissue), thereby creating new possibilities for monitoring cancer treatment and therapy by allowing doctors to evaluate how well a patient is responding to treatment.
  • Antigens and antibodies: Microarrays can also be used for the detection of antigens, antibodies, cytokines, and other immune related biomarkers in serum or other bodily fluids and tissues for the detection of autoimmune disorders (diseases in which the body's immune system attacks itself), viral and bacterial infections, and food and other allergies.
  • Protein characterization: High-throughput protein microarray is a versatile tool for genetic analysis of proteins, as well as protein profiling and characterization, including determination of interactions with other proteins, nucleic acids, enzymes, small molecules and drugs for comparison in normal state and disease conditions. Such comparisons can help in understanding how diseases impair protein pathways, induce the production of abnormal proteins and chemicals, and how relevant proteins can be targeted for the treatment.
  • Drug development: Protein microarrays are widely used, especially in the development of novel drugs targeting particular proteins and pathways, optimizing existing drugs, and understanding drug responses in terms of interactions, cross-reactivity, and toxicity.

  • Preparation of capture molecules and protein probes is currently limited, as only a small number of appropriate proteins and antibodies have been identified and are currently available for use, although research continues to expand selection.
  • Certain proteins may be insufficiently stable or may change the structure and function of target proteins upon interaction with some microarray platforms or surfaces, and as such may not be suitable for use in certain contexts.
  • Some microarrays may also interfere with certain image analysis techniques due to high levels of background noise. This noise is often due to nonspecific binding of sample proteins to the chemical coating on the surface of the array. Such noise may be reduced to a certain extent by tailoring the coating of the array, and also by increasing the signal intensity of specific binding proteins.
  • Some fluorescent labels used for detection may alter the probe's ability to interact with target proteins, thereby impairing protein interactions on the microarray and interfering with results. This may be improved by using fluorescent tags that do not interfere with targeted protein interactions.


Future research
  • Research is currently under way to develop protein libraries for a variety of protein microarray studies and analyses, as very limited sets of characterized proteins are available. Protein libraries are collections of proteins that have been identified and their structure, function, and interactions with other molecules or substances characterized.
  • Microarray detection methods are also being modified to increase the sensitivity and specificity of their detection capabilities. For example, fluorescent readout sensitivity can be increased 10-100 fold by a technique called tyramide signal amplification (TSA), which magnifies fluorescent markers. Label-free detection methods, such as carbon nanotubes, carbon nanowires, and microelectromechanical systems cantilevers, are other advances which are in their infancy but offer great promise. These label-free detection methods produce less background noise as they prevent nonspecific binding of sample proteins with the chemical coating of the surface of the array.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (

  1. Affymerix® . Accessed on July 29, 2008.
  2. Cekaite L, Hovig E, Sioud M. Protein arrays: a versatile toolbox for target identification and monitoring of patient immune responses. Methods Mol Biol. 2007;360:335-48.
  3. Hall DA, Ptacek J, Snyder M. Protein microarray technology. Mech Ageing Dev. 2007 Jan;128(1):161-7. Epub 2006 Nov 28.
  4. Mezzasoma L,Bacarese-Hamilton T, Di Christina M, et al. Antigen microarrays for serodiagnosis of infectious diseases. Clin Chem. 2002 Jan;48(1):121-30.
  5. Molecular Station: Bioinformatics, Protocols, DNA RNA Protein Proteomics.
  6. Natural Standard: The Authority on Integrative Medicine.
  7. Ramachandran N, Larson DN, Stark PR, et al. Emerging tools for real-time label-free detection of interactions on functional protein microarrays. FEBS J. 2005. Nov;272(21):5412-25.
  8. Salcius M, Michaud GA, Schweitzer B, et al. Identification of small molecule targets on functional protein microarrays. Methods Mol Biol. 2007;382:239-48.
  9. Stoll D, Templin MF, Bachmann J, et al. Protein microarrays: applications and future challenges. Curr Opin Drug Discov Devel. 2005 Mar;8(2):239-52.
  10. TeleChem International Inc. ArrayIt® Microarray Technology.
  11. Zhu X, Gerstein M, Snyder M. ProCAT: a data analysis approach for protein microarrays. Genome Biol. 2006;7(11):R110.

Copyright © 2011 Natural Standard (

The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

Search Site