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.
In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.
Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by electrophoresis using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.
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 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.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
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.
In molecular biology and genetics, a blot is a method of transferring large biomolecules onto a carrier, such as a membrane composed of nitrocellulose, polyvinylidene fluoride or nylon. In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred molecules are then visualized by colorant staining, autoradiographic visualization of radiolabelled molecules, or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity is visualized by incubation with a proper reagent, rendering either a colored deposit on the blot or a chemiluminescent reaction which is registered by photographic film.
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins. Computational methods typically use computer programs to analyze proteins. However, many experimental methods require computational analysis of the raw data.
An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA gang replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin and Fried and Crothers.
The far-western blot, or far-western blotting, is a molecular biological method based on the technique of western blot to detect protein-protein interaction in vitro. Whereas western blot uses an antibody probe to detect a protein of interest, far-western blot uses a non-antibody probe which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is employed to detect protein/protein interactions.
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.
Electroblotting is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands like lectins, or stains. This method can be used with all polyacrylamide and agarose gels. An alternative technique for transferring proteins from a gel is capillary blotting.
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.
The northwestern blot, also known as the northwestern assay, is a hybrid analytical technique of the western blot and the northern blot, and is used in molecular biology to detect interactions between RNA and proteins. A related technique, the western blot, is used to detect a protein of interest that involves transferring proteins that are separated by gel electrophoresis onto a nitrocellulose membrane. A colored precipitate clusters along the band on the membrane containing a particular target protein. A northern blot is a similar analytical technique that, instead of detecting a protein of interest, is used to study gene expression by detection of RNA on a similar membrane. The northwestern blot combines the two techniques, and specifically involves the identification of labeled RNA that interact with proteins that are immobilized on a similar nitrocellulose membrane.
A blotting matrix, in molecular biology and genetics, is the substrate onto which macromolecules, such as proteins, are transferred in a blot method. The matrices are generally chemically modified paper filters or microporous membrane filters. In a dot blot, macromolecules are applied directly to the matrix. Macromolecules can also be separated and transferred via gel electrophoresis.
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.
