Azure Biosystems
Arrays and microarrays offer a way to carry out hundreds, thousands, and even tens of thousands of miniature hybridization assays in parallel. Interactions between many types of molecules can be studied in an array format. Regardless of the specific interaction probed, the theory behind all array experiments is similar: a selection of samples is spotted in a grid pattern on a substrate, such as a PVDF membrane or glass slide, then incubated with another sample to determine whether components of that sample interact with or bind to the molecules on the slide. Depending on the experimental design, the bound molecules are detected by phosphor imaging, chemiluminescence, or fluorescence.
DNA microarrays consist of probes – fragments of known nucleotide sequences – spotted on glass slides. Labeled sample DNA hybridizes to the probes based on sequence homology. This interaction reveals whether a sequence of interest is present in a sample. DNA microarrays are frequently used to assess gene expression. For gene expression analysis, mRNA from a sample is transcribed into cDNA, labeled with a fluorophore, and incubated with the slide so any complementary cDNA can bind to the matching probe on the slide. Unbound cDNA is washed away, and the slide is imaged to see which cDNAs in the sample were homologous to and bound to probes on the array. Typically, the array is incubated simultaneously with two samples that have been labeled with two different dyes. For example, the array may be incubated with a control sample labeled with Cy3 and a test sample labeled with Cy5. This allows the relative expression of each gene to be compared between samples within the same experiment, inherently controlling for any irregularities in spot size or concentration that can occur between arrays.
Protein arrays include a variety of designs, from dot blots containing dozens of proteins, to membrane protein arrays containing hundreds of proteins, to protein microarrays that can assess thousands of proteins simultaneously. Several different approaches have been developed for studying proteins using arrays. Functional protein arrays involve spotting known purified proteins on a slide and assessing which proteins, small molecules, or nucleotides in a sample bind to the spotted proteins. These functional protein arrays are a way to probe protein-protein and/or protein-substrate interactions. In some experimental approaches, the protein array is incubated with enzyme substrates to determine which proteins on the array have enzymatic activity or which substrates can be metabolized by known enzymes. In contrast, analytical protein arrays are used to detect whether a certain protein is present in a sample.
Analytical protein arrays have known capture probes (often specific antibodies) bound to the slide. A sample is then incubated with the array to determine whether a protein of interest is present. The proteins may be labeled and detected in a variety of ways. Similar to Western blot detection, the sample proteins may be directly labeled with a fluorophore or radiolabel, or they may be detected indirectly using a secondary antibody which is labeled with a fluorophore or HRP. Depending on the spot size and detection chemistry, protein arrays can be imaged using a microarray scanner, a chemiluminescence imager, phosphor imaging, or X-ray film.
The array approach has been applied to other types of hybridization experiments as well. In lipid arrays, lipids are spotted on a membrane and incubated with a sample to determine whether proteins in the sample bind the spotted lipids. Reverse-phase protein arrays are essentially the opposite of antibody arrays. In reverse-phase protein arrays, cell lysates or other complex samples are spotted on the slide and the array is probed with antibodies to determine if specific proteins or modified forms of proteins are present in each sample, or to see if expression levels of proteins of interest differ between samples.
Arrays and microarrays allow the high-throughput processing and analysis of hybridization experiments. Arrays make it possible to interrogate thousands of potential interactions to screen drugs, identify protein-protein interactions, study gene expression, and more.
The Sapphire FL can capture images with a resolution as high as 5 microns, allowing it to image membrane arrays and some microarrays. With the ability to conduct fluorescence, chemiluminescence, and phosphor imaging, the Sapphire FL is compatible of a variety of array detection chemistries.
The Azure Imagers are also excellent choices to image membrane arrays detected using chemiluminescence or fluorescence. Using lasers for NIR fluorescent imaging sets the Azure Imagers apart from competitors, as our Imagers are the only ones on the market to use lasers. Narrow, specific laser excitation leads to higher signal, with less background and increases the overall sensitivity of your arrays and Western blots.
Having analysis software is also important for visualizing and analyzing microarray data and eventually facilitating data analysis for large experiments. For analysis, we recommend AzureSpot Pro software, which contains modules that can assist with annotating and analyzing images of blots and arrays. Request a free trial by clicking here.
Arrays and microarrays can be imaged using many different approaches depending on the spot sizes on the array and the detection chemistry. Some arrays may be imaged using fluorescent scanners, chemiluminescence imaging, or X-ray film.
DNA microarrays are often used for gene expression analysis. With DNA microarrays, it is possible to see whether the mRNA for a specific gene is present in a sample, and to quantify how much of the mRNA is present compared to another sample or standard.
Protein arrays contain tens to thousands of proteins spotted on a membrane or glass slide and are used to identify components that can interact with the spotted proteins. A sample is incubated with the protein array and the array is imaged to detect whether components of the sample, such as other proteins, nucleic acids, or small molecules, are able to interact with the proteins spotted on the array.
Compatible with a variety of array detection chemistries and images up to 5 microns
In this paper, Jobin et al evaluated which area of γ-aminobutyric acid type B (GABAB) receptor binds to Filamin A using a peptide array imaged on the Azure 400 Imager.
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ELISA (Enzyme-linked immunosorbent assay) and Western blotting are two of the most commonly used techniques in molecular biology for the detection and quantification of proteins.
If you’re reading this blog there’s a high chance you’re using antibodies in your work, in some form of immunoassay. However, many of us rarely
