Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
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.
Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.
The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.
The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.
Gel electrophoresis of nucleic acids is an analytical technique to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules are placed on a gel, where an electric field induces the nucleic acids to migrate toward the positively charged anode. The molecules separate as they travel through the gel based on the each molecule's size and shape. Longer molecules move more slowly because they the gel resists their movement more forcefully than it resists shorter molecules. After some time, the electricity is turned off and the positions of the different molecules are analyzed.
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, tests have shown it to have low mutagenicity without metabolic activation.
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely agarose or polyacrylamide. Variants of gel electrophoresis include SDS-PAGE, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting or immunoblotting to give additional information about a specific protein.
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies, reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis, crossed immunoelectrophoresis, rocket-immunoelectrophoresis, fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen.
A dot blot is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Instead, the sample is applied directly on a membrane in a single spot, and the blotting procedure is performed.
The single cell gel electrophoresis assay is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
An electrophoretic color marker is a chemical used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. The color markers are made up of a mixture of dyes that migrate through the gel matrix alongside the sample of interest. They are typically designed to have different mobilities from the sample components and to generate colored bands that can be used to assess the migration and separation of sample components.
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.
The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, eastern blot can be considered an extension of the biochemical technique of western blot. Multiple techniques have been described by the term "eastern blot(ting)", most use phosphoprotein blotted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel on to a polyvinylidene fluoride or nitrocellulose membrane. Transferred proteins are analyzed for post-translational modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the post-translational modifications and the probe, distinguishing them from a standard far-western blot. In principle, eastern blotting is similar to lectin blotting.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Epicocconone is a long Stokes' shift fluorogenic natural product found in the fungus Epicoccum nigrum. Though weakly fluorescent in water it reacts covalently yet reversibly with primary amines such as those in proteins to yield a product with a strong orange-red emission (610 nm). Epicoconone is notable because it the first covalent/reversible/turn-on fluorophore to be discovered and is a natural product with a new fluorescent scaffold. It is also cell membrane permeable, unlike many other fluorophores, and subsequently can be used in in vivo applications. Additionally, this dye can be used as a sensitive total protein stain for 1D and 2D electrophoresis, quantitative determination of protein concentration, making it a powerful loading control for Western blots.
Normalization of Western blot data is an analytical step that is performed to compare the relative abundance of a specific protein across the lanes of a blot or gel under diverse experimental treatments, or across tissues or developmental stages. The overall goal of normalization is to minimize effects arising from variations in experimental errors, such as inconsistent sample preparation, unequal sample loading across gel lanes, or uneven protein transfer, which can compromise the conclusions that can be obtained from Western blot data. Currently, there are two methods for normalizing Western blot data: (i) housekeeping protein normalization and (ii) total protein normalization.
SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.
