Enzymes are the catalysts for biochemical reactions. As such, they speed up the transition from reactants to products without being consumed in the process. Multiple enzymes can be found in every cell, from bacteria up through to humans. We as humans have found ways to exploit enzymes to produce food products, fuel, pharmaceutical products, biotechnological tools, sensors, and much more. The potential uses for enzymes are seemingly limitless. The creation of solid support structures with immobilized enzymes has improved our ability to reuse enzymes in a controlled manner for a variety of applications. Immobilized enzymes can be reused multiple times before their efficacy is lost. This allows them to be an affordable part of industrial processes.
Pharmaceutical validation is important to the manufacturing process to ensure product consistency and safety. It involves regulation of all raw materials and production procedures as well as testing of final product. The general rule of thumb is to follow good manufacturing practice (GMP). This demands that all protocols be up to date and followed by trained personnel. It also requires that equipment be well-maintained and inspected. In the case of clean-room usage the clean room needs to be verified.
An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. The assay can be used to identify the presence of pathogens in a clinical sample, or it can be used to measure the amount of a target biomolecule. If the goal of the immunoassay is to isolate a specific molecule then a separation system is needed. When the isolation is achieved by magnetic separation using a magnetic particle it is called a magneto-actuated immunoassay. The most common particle used in these assays is made of a core of magnetite that is coated with a biologically compatible material, and chemically modified by the attachment of antibodies. However, before designing a magnetic particle for an immunoassay one must decide which types of immunoassays best fits the goals of the experiment.
Superparamagnetism is a type of magnetism that lies between that of a permanent magnet and a paramagnet. Recall that a permanent magnet is always magnetic at temperatures below its Curie Temperature even in zero applied magnetic field, whereas a paramagnet is not magnetic at zero applied field but can become magnetic when an external magnetic field is applied. The potential for a paramagnet to be induced to have magnetization is called magnetic susceptibility. A superparamagnet behaves similarly to a paramagnet. The “super” means that it has a higher magnetic susceptibility than a regular paramagnet when a magnetic field is applied. Superparamagnets are typically made of iron oxide or other ferrous materials, and they are extremely small, on the order of 10-100 nanometers.
In bacteria there are two main types of DNA—genomic and plasmid. Plasmid DNA is unique to bacteria. Eukaryotic cells don't typically have plasmid DNA unless it was put there by transfection for experimental purposes. The most important goal when isolating nucleic acids is to obtain the highest purity genetic material possible. When isolating genomic DNA it is important to remove plasmid DNA and RNA from the sample. Similarly, sometimes an experiment calls for the isolation of plasmid DNA, and the selective removal of genomic DNA is necessary. Also, some commercial RNA isolation kits include gDNA eliminator spin columns to remove genomic DNA from the isolate.
Traditional immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are able to measure the presence or absence of only one analyte per reaction. Multiplex immunoassays measure dozens of different analytes in a single reaction. This is particularly beneficial for precious samples, and when only a small volume is collected for analysis. The multiplex immunoassay also saves working time since multiple assays can be completed simultaneously.
The ability to isolate and identify proteins from a biological solution is fundamental to basic research and clinical diagnosis. Proteins are the workhorses of the organism; they send and receive messages, they control the flow of information across the cell membrane, and they enact cascades of action within cells. It is rare that a single protein works alone, so it is imperative to understand how proteins interact with each other if we are to understand the nature of our bodies and to discover and treat disease.
Immunoprecipitation (IP) is a method used to purify target proteins from whole tissue or from cell culture. There are different types of IP: Single protein, Co-immunoprecipitation (co-ip), Chromatin immunoprecipitation(CHIP), RNA immunoprecipitation (RIP), and tagged protein immunoprecipitation. All of these techniques play an important role in modern medicine and research into the discovery of biochemical mechanisms, and they all are based on the basic principle of the specific lock-and-key interaction between antibody and antigen. Antibodies are naturally produced by the adaptive immune system, but they can also be engineered by modern techniques to specifically bind to any protein target desired.
Enzymes are used in the food, agricultural, cosmetic, and pharmaceutical industries to control and speed up reactions in order to quickly and accurately obtain a valuable final product. Enzymes are crucial to making cheese, brewing beer, baking bread, extracting fruit juice, tanning leather, and much more. The industrial uses of enzymes are also increasing since they are being used in the production of biofuels and biopolymers. The enzymes can be harvested from microbial sources or can be made synthetically. Yeast and E. coli are commonly engineered to overexpress an enzyme of interest. This type of enzyme engineering is a powerful way to obtain large amounts of enzyme for biocatalysis in order to replace traditional chemical processes.
Nano gold is another name for gold nanoparticles. These nanoparticles are a fraction of the size of human hair and are less than 100 nm in diameter. Nano gold particles are so small that it they are generally found as a colloidal solution, which means that the gold nanoparticles are suspended in a liquid buffer. Therefore, nano gold, or gold nanoparticles are also called colloidal gold. Also, nano gold is generally found in a colloidal solution because gold nanoparticles are created by citrate synthesis. This process involves mixing solutions together to result in the precipitation of gold nanoparticles into solution.
The force of gravity will cause sedimentation of particles from a heterogeneous mixture; larger and denser particles sedimentfaster than the smaller and less dense particles. This phenomenon is useful for separating heterogeneous solutions into independent components, and for the isolation and enrichment of target molecules, cells, and cell organelles. Differential centrifugation accelerates the separation process by introducing centripetal forces many times greater than gravity. The precipitated particles form a pellet at the bottom of the tube during centrifugation. The rate of sedimentation is dependent on the size and density of the particles, so centrifugation can be used to isolate target particles simply by controlling centrifugal force or the rate of centrifugation. The rate of centrifugation is reported as angular velocity by revolutions per minute (rpm) or as acceleration(g). RPM is dependent on the radius of the rotor in the centrifuge.