A CHIP assay is short for Chromatin Immunoprecipitation assay. This technique is used to gain insight into the region of the genome a particular protein is associated with. To do this the CHIP assay captures information about the protein-DNA interactions in the histones of genomic DNA. The protein-DNA binding structure is preserved because the histone is crosslinked before the cell is lysed. The protein and DNA are bound together even after the chromatin is broken up into smaller pieces. Then the protein-DNA conjugates are captured by immunoprecipitationwith antibodies binding specifically to regions in the protein of interest. Then the DNA is extracted from the proteins and analyzed by targeted qPCR or genome-wide next-generation sequencing (NGS).
Biotinylation is the process of attaching a biotin tag to a molecule. The molecule can be a protein or an oligonucleotide. Biotinylated molecules are useful in many biological contexts, but are primarily used for capture or detection of target molecules. Biotinylated molecules are used in Western blotting, ELISA, flow cytometry, and other inventive detection methods.
Filtration is a common technique for mechanically separatinggases or liquids. A filter membrane serves as a barrier to enable ion exchange, removing macromolecules or bacteria from solution, separating colloids, or recovering gases. Some uses for filtration include producing clean drinking water, generating safe food products, and ensuring a clean environment. It is used for petro-chemical vapor recovery, oxygenation of blood in an artificial lung, and hemodialysis via an artificial kidney. Filter membranes are necessary for ion exchange in a fuel cell and for electrolysis. Filtration is also a key procedure in the laboratory to separate RNA, DNA, cells, proteins, and other molecules.
Common methods include microfiltration (0.1 to 1 micrometer particulates), ultrafiltration (0.001 to 0.1 micron), nanofiltration (less than 0.001 micron), and reverse osmosis (less than 0.001 micron). Microfiltration is used to separate colloidal solutions, but the other three are used for dissolved particulates.
Enzyme immobilization onto solid supports is a convenient way to control and reuse enzymes. The enzyme is a catalyst for a reaction, meaning that it enables or speeds up the chemical conversion from reactants to products. This is a valuable property for both small scale research and large scale industry including biotechnology, pharamaceutical applications, food production, and wastewater treatment. There are many materials used for immobilized enzyme solid support systems, and a variety of enzymes can be used. Perhaps the most advantages solid support system is one that is magnetic. A magnetic bead is easily recoverable with the application of an external magnetic field. This ensures that reusing the enzyme is quick and easy.
The traditional biomagnetic separation is a permanent magnet block. The test tube is placed next to the magnet and the magnetic particles in solution move toward the magnet. This system works for very small volumes, but it is not the most efficient method and problems often arise in larger volumes. The downfall of this geometry is that the permanent magnet is only on one side of the tube, which means that the magnetic particles are only drawn to that one side. The magnetic particles close to the magnet will experience a higher force than the magnetic particles farthest away from the magnet. Magnetic force decreases as distance from the magnet increases, so the particles farthest away might not feel any force if the magnetic strength is not great enough.
Centrifugation is a common technique used for the separation of heterogeneous mixtures. The force of gravity on matter is the core principle of centrifugation. Matter naturally separates based on density, with the most dense particles precipitating out of solution first, and the less dense particles falling out later. Some particles prefer to remain in colloidal solution under normal gravitational force, and will not separate naturally within a reasonable time frame.Centrifugation can be used to rapidly separate those mixtures. The spinning centrifuge creates centripetal forces much greater than gravity, which can force particles to separate. The centrifugal protocol must be optimized for each experiment in order to efficiently separate the mixtures into desired layers. The most basic separation results in a higher density pellet at the bottom of the tube, and a lower density supernatant. Differential centrifugation is a process whereby the pellet and supernatant are separated during multiple centrifugation steps. Density gradient centrifugation utilizes a density gradient matrix within the tube to aid in separation. Another useful tool for centrifugation is a centrifugal filter. The filter helps to separate particles by size as well as density in one swift motion. The centrifugal filter is used to isolate RNA or DNA, to consolidate proteins, to separate molecules by size, or to remove contaminants from a liquid.
The reversible-binding systems mentioned in the previous post are examples of non-specific capture methods. They capture total DNA and RNA in a sample because they simply rely on the affinity of nucleic acids to the magnetic particle coating or functional moiety. Non-specific capture methods bind all single-strand (ssDNA) or RNA regardless of sequence. A more specific capture system is able to target specific sequences of ssDNA or RNA. This type of specific capture is most applicable to diagnostic systems as an assay for a specific pathogen. Examples include systems to diagnose methicillin resistant staphyloccocus aureaus (MRSA), specific plant viruses, and infection with malaria-causing bacteria. These strategies could be applied to detect viral, bacterial, or fungal pathogens in a variety of clinical samples.
Cleaning validation is an important regulatory component of industrial manufacturing. It ensures that a standard and reliable protocol is followed forthe removal of residual chemicals from equipment between batches. Clean equipment is important for process validation because consistent products cannot be synthesized without consistent starting points. However, cleaning validation is about more than just equipment cleaning. Attention must be paid to other parts of the manufacturing process including employee education, building and facility maintenance, equipment calibration and maintenance, control of raw materials, up-to-date standard operating procedures, clear communication between all employees, and accurate reporting of data and processes.
The coating and surface chemistry of magnetic beads governs the binding efficiency of target DNA or RNA. Superparamagnetic beads for life science applications come in two general forms: core-shell type and embedded type. The core-shell synthesis method produces beads composed of a single superparamagnetic core with a polymer or silica surface coating. One example is a bead composed of a magnetite core surrounded with a dextran shell. Other beads of the core-shell type are composed of a polystyrene or polyvinyl alcohol (PVA) core surrounded by superparamagnetic particles and protected by a surface coating.
Superbugs, or bacteria that are resistant to currently available antibiotic treatments, are of growing concern to human health worldwide. Many of these superbugs are present in hospitals, and are frequently colonizing surgical sites and causing life-threatening infections and sepsis. The presence of these bacteria in blood is commonly detected by traditional culture methods that require one to two days. There is a need for a faster method to identify the presence of the bacteria and to take measures to prevent the spread of infection as rapidly as possible. One proposed technology is to use immunomagnetic separation with conjugated fluorescent probes to selectively bind bacteria and quickly visualize their presence in whole blood samples. Recently, fluorescent magnetic multifunctional carbon dots have been coupled with superbug-specific antibodies for the identification of the drug-resistant superbugs Staphylococcus aureus (MRSA) and Salmonella enterica serotype typhimurium definitive phage type 104 (DT 104) present in whole blood samples.