Microarrays are the latest technological tools in gene identification studies. There are different microarrays like DNA microarray, Protein microarrays and antibody microarrays.
Protein microarrays are especially suited for identification of protein-protein interactions, protein-phospholipids interactions, protein- DNA and protein-ligand interactions etc. These arrays have found their application in clinical diagnostics and for monitoring disease progress.
Why Protein microarrays?
DNA microarrays are limited in their functionality since they convey information on only genes. They seldom provide sufficient information about the proteins which they encode. The study of proteins have been since been made through different technologies such as mass spectroscopy, tagging, sub cellular localization etc. The recent approach of protein microarrays have been a valuable development in the field.
They are similar to DNA microarrays and are available as plate, probes or attachments.
Types of protein microarrays
There are mainly three types
a. Analytical microarrays
b. Functional microarrays
c. Reverse phase microarrays
I. Analytical microarrays are used to identify and classify the different proteins in a mixture and measure the parameters such as binding affinity, specificity, protein expression levels.
It uses antibodies, affibodies and aptamers arrayed on a glass microscope slide. This is probed with protein solution. Antibody microarrays are the commonest among them.
Antibodies are used standard which are usually 150kDa. Affibodies are based on the protein A from Staph. aureus. They are smaller than antibodies with alpha helical structures. Aptamers are small sequences of DNA or RNA which have variable loops in their structure and specific folding patterns due to such regions. They bind to protein and target molecules by Van der Waals forces, Hydrogen bonding and electrostatic interactions. Their high specificity enables them to functions as probe molecules and these can be synthesized in the laboratory and selected using in vitro techniques.
The arrays are prepared either on glass or silicon plates. The probes are attached by
• Covalent binding with amines
• Adsorption to nitrocellulose, poly Lysine, Acrylamide etc.
• Affinity binding - Streptavidin and Biotin
Analytical protein microarrays using aptamers which are able to recognize drug-resistant HIV-1 reverse transcriptase are used in developing personalized medicine. Neutravidin-biotin affinity binding is used. The technique usually employs two antibodies, primary and secondary.
Primary antbodies are not marked with the fluorescent dyes and secondary antibodies which bind on to the surface of primary antibodies are marked with Cy-3 labelling. The primary antibody binds to the array first followed by secondary antibodies which bind to the surface of primary.
The hybridization efficiency is calculated in the usual process of scanning, quantitiation, normalization and analysis.
II. Functional protein microarrays have arrays which have protein domains or functional proteins. This is used to analyze the functions of proteins and biochemical activities of the entire proteome. These are also effective in studying protein-protein, protein-DNA, protein-phospholipid, and protein- ligand interactions.
The samples used for functional microarray are purified and labeled molecules of nucleic acids, proteins, lipids and other ligands.
These chips are also efficient in studying protein-drug interactions and binding between enzyme and substrate.
III. The third and final type of protein microarray is the Reverse phase protein microarray. In this RPA method, the cells once isolated from the specimen tissues are lysed using various enzymes. This is them used to coat the nitrocellulose slide. The probes are usually antibodies which can specifically bind to target protein. Detection is usually done by chemiluminescence, fluorescence, and colorimetric assays. Proteins can be quantified using reference peptides.
The RPA is used to determine the presence of disease, altered proteins due to post translational modifications etc. This can then be used to design unique therapies which target such proteins.
The technique is highly sensitive and exhibit good reproducibility with experiments employing antibodies. This has found many applications in personalized medicine and developing diagnostics.
Down regulation of genes involved in MAPK pathway activation was found in colorectal cancer using the RPA technology.
Chip formats used in protein microarray
There are mainly two types of formats used in protein microarrays- glass slides and nanowells. The method chosen should
• retain proteins in an active state even at higher densities
• should be compatible with the available scanners and printers
• proteins should be kept in a moisturized environment.
• Compatible with standard equipments used DNA microarray experiments.
• Cost less
• Susceptible to cross contamination
• Protein solution can get evaporated faster.
Nanowell/ Mcrowell chips
• Suitable for assys with multiple components
• Effective in solution based assays
• Less evaporation rate
• Absence of cross contamination
• Costs less
• Disposable silicone or poly dimethyl siloxane is used for microwells. GPTS (3-glycidoxypropyltrimethoxysilane) is used as a cross linking agent.
• Special equipments are needed to fill the nanowells with higher densities of protein solution.
• High end analysis can be performed with mass spectrometry and surface plasmon resonance analysis with a little modification of the wells.
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