Microarray technology can simultaneously analyze thousands of parameters in a single experiment. Micro-point of capture molecules are fixed into ranks on a solid support and exposed to samples containing corresponding binding molecules. Complex formation in each micro-point can be detected by the readout system, which is based on fluorescence, chemiluminescence, mass spectrometry, radioactive or electrochemistry. Miniaturization and parallelization binding assays, whose analysis power can be also enlarged by microarray gene expression analysis, is sensitive. These systems can be used to detect the degree of hybridization and immobilized DNA microarray probes will be exposed to complementary target. Currently, the development of protein array has demonstrated its applications in enzyme-substrate, DNA- protein and different types of protein - protein interactions. In this post, we will discuss the capture-molecule-ligand analysis, analyze its theoretical advantages and disadvantage and its influence in diagnostics, genomic and proteomics.
Theoretically, any kind of ligand-binding assay, which is depending on the product formation of immobilized capture molecules and the target existing in the surrounding solution can be miniaturized and parallelized. This process can be performed in microarray. The nucleic acid - nucleic acid interaction, which is also called DNA chips, has been well established, while protein array analysis just begins. This can be reflected in recent nucleic acid - protein, protein - protein, ligand - receptor and enzyme - substrate microarray analysis articles.
It is Bulyk and his colleagues who found the application of microarray in DNA- protein interaction. They created the double-stranded oligonucleotide microarray. High concentrations of single-stranded oligonucleotide microarray is produced by using Affymetrix (Santa Clara, CA, USA) technology. Single-stranded oligonucleotide microarray was converted into a double-stranded oligonucleotide microarrays under the influence of enzymes extension reaction. Generally speaking, DNA- protein interaction analysis is useful for characterizing and identifying DNA-binding proteins, such as, transcription factors.
There are many kinds of enzyme to analyze enzyme-substrate. In a conceptual proof experiments, MacBeath and Schreiber fixed three types of kinase substrate on planar glass surface. Each microarray, which corresponds to an individual kinase, incubates with radioactively labeled ATP. Each substrate can only be phosphorylated by its specific kinase. In an advanced experiment, Zhu and his colleagues detected the activity of 119 different protein kinases, which is from the Saccharomyces cerevisiae, in 17 different substrates. They used a plate with microwells, in which the substrate interacted. Kinase, which is expressed into GST fusion proteins, is incubated with substrate and radiolabeled ATP in microwells. After kinase reaction, the kinase and ATP will be washed away, while the array uses phosphateimager to analyze the phosphorylated substrate. The new activity of single kinase can be identified with this method.
As for receptor - ligand analysis, the small organic molecules produced by the solid phase chemistry combination were immobilized in a microarray. The single resin beads from combinatorial synthesis are placed in 96-well plates, from which organic molecules are released chemically. Organic molecules are diluted, dispersed into small dots, and covalently attached on the slide. These target protein microarrays generated by the so-called small-molecules printing are incubated with the target protein with fluorescently labeled.
In the field of protein - protein interaction analysis, dot-blot filter analysis is used for screening the interactions between immobilized protein specific and other proteins. Some interactions, which happens between radioactively labeled human p52 GST fusion proteins and immobilized capture proteins, have been detected, such as, nuclear proteins, serine - arginine protein fragments isolated from HeLa cells. What’s more, this technology has revealed the DNA, RNA, or the interaction between low molecular weight ligands with immobilized molecules.
Recent work of Zhu and his colleages proved the great power of microarray in proteomic field. After the purification of 5800 different kinds of recombinant proteins from S. Cerevisiae, the complex protein arrays containing 90 percent of the microbial gene were generated. These protein arrays can be used to study the whole- genome-wide protein-protein interactions. Using calmodulin as model protein to probe arrays can confirm a number of known interactions and measure a range of novel binding proteins. Experiment detecting protein - lipid interaction convincingly explains the possibility of detecting the protein, which can bind with low-molecular-weight complexes.
We use microarray technology to screen antigen - antibody interactions. Autoimmune diseases, such as, system rheumatism, can be diagnosed with 18 different antigens, which were immobilized in a microarray. In 1 ml serum in patients, we can detect the high accuracy antibody titers. Sandwich immunoassay is also miniaturized and parallelled, and may be done in microarray. This has been proved by the different levels of cytokines in biological samples. This will ultimately lead to powerful and reliable diagnostic analysis.
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