|
Blotting
In molecular biology and genetics, a blot is a method of transferring large biomolecules (proteins, DNA or RNA) 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 (for example, silver staining of proteins), autoradiographic visualization of radiolabelled molecules (performed before the blot), 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 (and so, the molecules found in the blot) is visualized by incubation with a proper reagent, rendering either a colored deposit on th ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Eastern Blot
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, easter ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Northern Blot
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.Kevil, C. G., Walsh, L., Laroux, F. S., Kalogeris, T., Grisham, M. B., Alexander, J. S. (1997) An Improved, Rapid Northern Protocol. Biochem. and Biophys. Research Comm. 238:277–279. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression rates during differentiation and morphogenesis, as well as in abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. Strictly speaking, the term 'northern blot' refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting m ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Western Blot
The western blot (sometimes called the protein immunoblot), 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. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibod ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Blotting Compass For Molecular Probes
In molecular biology and genetics, a blot is a method of transferring large biomolecules (proteins, DNA or RNA) 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 matrix, blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred molecules are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive tracer, radiolabelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibody, 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 (and so, the molecules found in the blot) is visualized by incubation with a proper reag ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Molecular Biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactions. Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury, who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observ ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Southwestern Blot
The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated. Southwestern blotting was first described by Brian Bowen, Jay Steinberg, U.K. Laemmli, and Harold Weint ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Southern Blot
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 (such as blood or tissue) 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. The Southern blotting combines the transfer of electrophoresis-separated DNA fragments to a filter membrane in a process called blotting, and the subsequent fragment detection by probe hybridization. ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Reverse Northern Blot
The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a tissue and radioactively labelled. A reverse northern blot can be used to profile expression levels of particular sets of RNA sequences in a tissue or to determine presence of a particular RNA sequence in a sample. Although DNA Microarrays and newer next-generation techniques have generally supplanted reverse northern blotting, it is still utilized today and provides a relatively cheap and easy means of defining expression of large sets of genes. Procedure In order to prepare the reverse northern membrane, cDNA sequences for transcripts of interest are immobilized on nylon membranes, which can be accomplished by use o ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Far-western Blot
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. Method In conventional western blot, gel electrophoresis is used to separate proteins from a sample; these proteins are then transferred to a membrane in a 'blotting' step. In a western blot, specific proteins are then identified using an antibody probe. Far-western blot employs non-antibody proteins to probe the protein of interest on the blot. In this way, binding partners of the probe (or the blotted) protein may be identified. The probe protein is often produced in '' E. coli'' using an expression clon ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Blotting Matrix
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. One of the most common blotting matrices for protein analysis is nitrocellulose, which has a high affinity for proteins due to hydrophobic interactions. However, proteins with low molecular weight have a small affinity for nitrocellulose, limiting potential applications. This defect may be remedied by glutaraldehyde, which can covalently bond proteins to nitrocellulose. Another matrix is cellulose paper modified with diazophenylthiother, which can also facilitate covalent bonding of proteins. Nylon membranes are also used for protein blotting, although they may result in the binding of anionic dyes suc ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Dot Blot
A dot blot (or slot 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 technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target protein. Uses Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost. Dot blots are also performed to screen the binding capabilities of an antibody. Methodology A general dot blot protocol involves spotting 1–2 microliters of a samples onto a nitrocellulose Nitrocellulose (also known as cellulose nitrate, flash paper, flash cotton, guncot ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Blotting Matrix
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. One of the most common blotting matrices for protein analysis is nitrocellulose, which has a high affinity for proteins due to hydrophobic interactions. However, proteins with low molecular weight have a small affinity for nitrocellulose, limiting potential applications. This defect may be remedied by glutaraldehyde, which can covalently bond proteins to nitrocellulose. Another matrix is cellulose paper modified with diazophenylthiother, which can also facilitate covalent bonding of proteins. Nylon membranes are also used for protein blotting, although they may result in the binding of anionic dyes suc ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
