Microarrays provide a way of organizing biological samples for high-throughput analysis. Samples are arranged in columns and rows upon a support surface consisting of a glass slide, a nitrocellulose membrane or a microtiter plate.
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Protein microarrays are used to determine the function of proteins, as well as to monitor their interactions. The structure of the array allows for numerous proteins to be tracked in parallel. Protein microarrays were developed by utilizing the technology of DNA microarrays, which are commonly used to analyze gene expression.
The requirements of protein microarrays are, however, more complex and necessitate material customization to make them suitable. Even though they are not as commonly seen as DNA microarrays, protein microarrays are increasingly being used for multiple applications in the biosciences.
Application for detecting protein interactions
One of the earliest applications of protein microarrays was for the detection of protein-binding properties. Test ligands are directly or indirectly labelled with fluorescent dyes to analyze protein-protein interactions. One of the proteins in the pair being tested is arrayed in multiple samples on slides and each sample is probed with a different fluorescently labelled protein. Interactions are identified in the sample spots where the fluorescent label is visible.
Protein microarrays have been utilized for monitoring the interactions between:
- Different proteins such as enzymes and substrates
- Proteins and antibodies
- Proteins and lipids
- Proteins and peptides
- Proteins and low-molecular mass compounds
- Proteins and oligosaccharides
- Proteins and DNA
- Proteins and RNA
- Lectin (carbohydrate-binding proteins) and cells
Application for clinical diagnostics and prognosis
Protein microarrays have also been applied for use in clinical diagnostics and prognosis. Biomarker identification tools can be formed when proteins are treated as potential antigens that can be tested for association with particular diseases.
For example, fluorescently labelled anti-human immunoglobulin antibodies are used to detect the pairings of antigens and antibodies. The autoantibody associated with a particular human disease will recognize the human protein sample on the array and the fluorescent label provides a method of identifying the pairing produced. This method creates a profile of autoantibodies associated with the disease.
A similar technique has been developed for the detection of infectious diseases. One example is the coronavirus protein microarray produced for diagnosing severe acute respiratory syndrome (SARS). The array included samples of SARS coronavirus proteins and five additional infectious coronaviruses.
The presence of the human antibodies against the SARS coronavirus proteins on the microarray led to the ability to distinguish patient serum samples that included the virus with 94% accuracy. In short, protein microarray technology provided a diagnostic method with at least one hundred times the sensitivity than standard diagnostic methods, along with lower sample volume requirements.
Application of the reverse phase protein microarray (RPPA)
Reverse phase protein microarrays (RPPA) provide a method for application on pure protein lysates isolated from tissue or cultured cells. The technology combines microarrays with laser capture microdissection (LCM).
LCM is a method of procuring cells using a laser capable of melting an area between 7.5 and 30 micrometers in diameter. The tissue is stained, placed on a slide then visualized under a microscope in real-time.
Cells can be isolated within the diameter of the laser, with more cells obtained by moving the slide. The isolated cells are then lysed in preparation for being placed on the reverse phase protein microarray. The sample spots on the array contain the proteins within a cell that correspond to the given pathological state.
While conventional protein arrays immobilize the antibody probe, the reverse phase protein microarray immobilizes the protein being analyzed. Reverse phase protein microarrays have been used for diagnostics, to investigate signal transduction pathways between hormones and the cell membrane, as well as for identifying potential vaccine candidates.
Reviewed by: Dr Tomislav Meštrović, MD, PhD