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A protein microarray is a piece of glass on which different molecules of protein have been affixed at separate locations in an ordered manner thus forming a microscopic array. These are used to identify protein-protein interactions, to identify the substrates of protein kinases, or to identify the targets of biologically active small molecules. The most common protein microarray is the antibody microarray, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysate solutions.
Additional recommended knowledge
Protein microarrays (also biochip, proteinchip) are measurement devices used in biomedical applications to determine the presence and/or amount (referred to as quantitation) of proteins in biological samples, e.g. blood. They have the potential to be an important tool for proteomics research. Usually a multitude of different capture agents, most frequently monoclonal antibodies, are deposited on a chip surface (glass or silicon) in a miniature array. This format is often also referred to as a microarray (a more general term for chip based biological measurement devices).
Types of chips
There are several types of protein chips, the most common being glass slide chips and nano-well arrays.
Production of protein arrays
The proteins can be externally synthesised, purified and attached to the array. Alternatively they can be synthesised in-situ and directly attached to the array.
The proteins can be synthesised through biosynthesis, cell-free DNA expression or chemical synthesis. In-situ synthesis is possible with the latter two. With cell-free DNA expression, proteins are attached to the support right after their production. Peptides chemically procured by solid phase peptide synthesis are already attached to the support. Selective deprotection is carried out through lithographic methods or by the so-called SPOT-synthesis.
Artifacts to Avoid
1) To avoid results variability make sure you've got a very efficient lysis of the buffer. Use consistent sample processing conditions; 2) Many antibodies don't work well as capture reagents, even if they do work well in western blotting and other denaturing conditions. Some antibodies often bind poorly to intact proteins in a cell extract; 3) Different proteins like different solution conditions, so if you do not see binding it doesn't mean that there is no binding between the two partners in physiological conditions; 4) Adjust the solute conditions to avoid non-specific association: change salt concentration, pH, add 1% alignate; 5) on the array's surface the conjugated protein should be in the right conformation (i.e., folded, NOT denatured), anchored by the same amino acid (in the same orientation), and be kept away from the surface by a linker to avoid steric hindrance.
Types of capture molecules
Capture molecules used are most commonly antibodies; however, more recently there has been a push towards other types of capture molecules which are more similar in their nature such as peptides or aptamers. Antibodies have several problems including the fact that there are not antibodies for most proteins and also problems with specificity in some commercial antibody preparations. Nevertheless, antibodies still represent the most well-characterized and effective protein capture agent for microarrays. Recently, nucleic acids, receptors, enzymes, and proteins have been spotted onto chips and used as capture molecules. This allows a vast variety of experiments to be conducted on protein-protein interactions, and all other protein binding substrates.
Although protein microarrays may use similar detection methods as DNA Microarrays, a problem is that protein concentrations in a biological sample may be many orders of magnitude different from that for mRNAs. Therefore, protein chip detection methods must have a much larger range of detection.
The preferred method of detection currently is fluorescence detection. Fluorescent detection is safe, sensitive, and can have a high resolution. The fluorescent detection method is compatible with standard microarray scanners, however some minor alterations to software may need to be made.
MACHARLA SHARATH KUMAR (WWW.SHARATH88080@YAHOO.CO.IMN
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Protein_microarray". A list of authors is available in Wikipedia.|