Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
The molecular mass is the mass of a given molecule. Units of daltons (Da) are often used. Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The derived quantity relative molecular mass is the unitless ratio of the mass of a molecule to the atomic mass constant.
Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their shape, and in some cases size. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
The lipid bilayer is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of a lipid bilayer, as are the nuclear membrane surrounding the cell nucleus, and membranes of the membrane-bound organelles in the cell. The lipid bilayer is the barrier that keeps ions, proteins and other molecules where they are needed and prevents them from diffusing into areas where they should not be. Lipid bilayers are ideally suited to this role, even though they are only a few nanometers in width, because they are impermeable to most water-soluble (hydrophilic) molecules. Bilayers are particularly impermeable to ions, which allows cells to regulate salt concentrations and pH by transporting ions across their membranes using proteins called ion pumps.
Semipermeable membrane is a type of synthetic or biologic, polymeric membrane that allows certain molecules or ions to pass through it by osmosis. The rate of passage depends on the pressure, concentration, and temperature of the molecules or solutes on either side, as well as the permeability of the membrane to each solute. Depending on the membrane and the solute, permeability may depend on solute size, solubility, properties, or chemistry. How the membrane is constructed to be selective in its permeability will determine the rate and the permeability. Many natural and synthetic materials which are rather thick are also semipermeable. One example of this is the thin film on the inside of an egg.
Ultrafiltration (UF) is a variety of membrane filtration in which forces such as pressure or concentration gradients lead to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate (filtrate). This separation process is used in industry and research for purifying and concentrating macromolecular (103–106 Da) solutions, especially protein solutions.
Passive transport is a type of membrane transport that does not require energy to move substances across cell membranes. Instead of using cellular energy, like active transport, passive transport relies on the second law of thermodynamics to drive the movement of substances across cell membranes. Fundamentally, substances follow Fick's first law, and move from an area of high concentration to an area of low concentration because this movement increases the entropy of the overall system. The rate of passive transport depends on the permeability of the cell membrane, which, in turn, depends on the organization and characteristics of the membrane lipids and proteins. The four main kinds of passive transport are simple diffusion, facilitated diffusion, filtration, and/or osmosis.
In molecular biology and pharmacology, a small molecule or micromolecule is a low molecular weight organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs are small molecules; the terms are equivalent in the literature. Larger structures such as nucleic acids and proteins, and many polysaccharides are not small molecules, although their constituent monomers are often considered small molecules. Small molecules may be used as research tools to probe biological function as well as leads in the development of new therapeutic agents. Some can inhibit a specific function of a protein or disrupt protein–protein interactions.
In chemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing.
Hemodialysis, also spelled haemodialysis, or simply dialysis, is a process of filtering the blood of a person whose kidneys are not working normally. This type of dialysis achieves the extracorporeal removal of waste products such as creatinine and urea and free water from the blood when the kidneys are in a state of kidney failure. Hemodialysis is one of three renal replacement therapies. An alternative method for extracorporeal separation of blood components such as plasma or cells is apheresis.
In cellular biology, membrane transport refers to the collection of mechanisms that regulate the passage of solutes such as ions and small molecules through biological membranes, which are lipid bilayers that contain proteins embedded in them. The regulation of passage through the membrane is due to selective membrane permeability – a characteristic of biological membranes which allows them to separate substances of distinct chemical nature. In other words, they can be permeable to certain substances but not to others.
An artificial membrane, or synthetic membrane, is a synthetically created membrane which is usually intended for separation purposes in laboratory or in industry. Synthetic membranes have been successfully used for small and large-scale industrial processes since the middle of the twentieth century. A wide variety of synthetic membranes is known. They can be produced from organic materials such as polymers and liquids, as well as inorganic materials. Most commercially utilized synthetic membranes in industry are made of polymeric structures. They can be classified based on their surface chemistry, bulk structure, morphology, and production method. The chemical and physical properties of synthetic membranes and separated particles as well as separation driving force define a particular membrane separation process. The most commonly used driving forces of a membrane process in industry are pressure and concentration gradient. The respective membrane process is therefore known as filtration. Synthetic membranes utilized in a separation process can be of different geometry and flow configurations. They can also be categorized based on their application and separation regime. The best known synthetic membrane separation processes include water purification, reverse osmosis, dehydrogenation of natural gas, removal of cell particles by microfiltration and ultrafiltration, removal of microorganisms from dairy products, and dialysis.
Gas mixtures can be effectively separated by synthetic membranes made from polymers such as polyamide or cellulose acetate, or from ceramic materials.
Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.
In ultrafiltration, the molecular weight cut-off or MWCO of a membrane refers to the lowest molecular weight of the solute for which 90% of the solute is retained by the membrane, or the molecular weight of the molecule that is 90% retained by the membrane.
Membrane technology encompasses the scientific processes used in the construction and application of membranes. Membranes are used to facilitate the transport or rejection of substances between mediums, and the mechanical separation of gas and liquid streams. In the simplest case, filtration is achieved when the pores of the membrane are smaller than the diameter of the undesired substance, such as a harmful microorganism. Membrane technology is commonly used in industries such as water treatment, chemical and metal processing, pharmaceuticals, biotechnology, the food industry, as well as the removal of environmental pollutants.
The cell membrane is a biological membrane that separates and protects the interior of a cell from the outside environment. The cell membrane consists of a lipid bilayer, made up of two layers of phospholipids with cholesterols interspersed between them, maintaining appropriate membrane fluidity at various temperatures. The membrane also contains membrane proteins, including integral proteins that span the membrane and serve as membrane transporters, and peripheral proteins that loosely attach to the outer (peripheral) side of the cell membrane, acting as enzymes to facilitate interaction with the cell's environment. Glycolipids embedded in the outer lipid layer serve a similar purpose.
Diafiltration is a dilution process that involves removal or separation of components of a solution based on their molecular size by using micro-molecule permeable filters in order to obtain pure solution.
Desalting and buffer exchange are methods to separate soluble macromolecules from smaller molecules (desalting) or replace the buffer system used for another one suitable for a downstream application. These methods are based on gel filtration chromatography, also called molecular sieve chromatography, which is a form of size-exclusion chromatography. Desalting and buffer exchange are two of the most common gel filtration chromatography applications, and they can be performed using the same resin.
