|
ICLIP
iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPrecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, which uses UV light to covalently bind proteins and RNA molecules to identify RNA binding sites of proteins. This crosslinking step has generally less background than standard RNA immunoprecipitation (RIP) protocols, because the covalent bond formed by UV light allows RNA to be fragmented, followed by stringent purification, and this also enables CLIP to identify the positions of protein-RNA interactions. As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes by stringent washing during immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. The labelled protein-RNA complexes are then visualised for quality control, excised from nitrocellulose, and treated with proteinase to release the RNA, leaving only a few amino acids at the crosslink site of th ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cross-linking Immunoprecipitation
Cross-linking immunoprecipitation (CLIP) is a method used in molecular biology that combines UV cross-linking with immunoprecipitation in order to analyse protein interactions with RNA or to precisely locate RNA modifications (e.g. m6A). CLIP-based techniques can be used to map RNA binding protein binding sites or RNA modification sites of interest on a genome-wide scale, thereby increasing the understanding of post-transcriptional regulatory networks. Workflow CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV). Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity. The cross-linked cells are then lysed, and the protein of interest is isolated via immunoprecipitation. In order to allow for sequence specific priming of reverse transcription, RNA adapters are ligated to the 3' ends, while radiolabeled phosphates are transferred to the 5' ends of the RNA fragments. The RNA-protein comple ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Intramembrane Protease
Intramembrane proteases (IMPs), also known as intramembrane-cleaving proteases (I-CLiPs), are enzymes that have the property of cleaving transmembrane domains of integral membrane proteins. All known intramembrane proteases are themselves integral membrane proteins with multiple transmembrane domains, and they have their active sites buried within the lipid bilayer of cellular membranes. Intramembrane proteases are responsible for proteolytic cleavage in the cell signaling process known as regulated intramembrane proteolysis (RIP). Intramembrane proteases are not evolutionarily related to classical soluble proteases, having evolved their catalytic sites by convergent evolution. Although only recently discovered, intramembrane proteases are of significant research interest because of their major biological functions and their relevance to human disease. Classification There are four groups of intramembrane proteases, distinguished by their catalytic mechanism: *Metalloproteases: ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
UV Light
Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30 PHz) to 400 nm (750 THz), shorter than that of visible light, but longer than X-rays. UV radiation is present in sunlight, and constitutes about 10% of the total electromagnetic radiation output from the Sun. It is also produced by electric arcs and specialized lights, such as mercury-vapor lamps, tanning lamps, and black lights. Although long-wavelength ultraviolet is not considered an ionizing radiation because its photons lack the energy to ionize atoms, it can cause chemical reactions and causes many substances to glow or fluoresce. Consequently, the chemical and biological effects of UV are greater than simple heating effects, and many practical applications of UV radiation derive from its interactions with organic molecules. Short-wave ultraviolet light damages DNA and sterilizes surfaces with which it comes into contact. For huma ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cross-link
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins). In polymer chemistry "cross-linking" usually refers to the use of cross-links to promote a change in the polymers' physical properties. When "crosslinking" is used in the biological field, it refers to the use of a probe to link proteins together to check for protein–protein interactions, as well as other creative cross-linking methodologies. Although the term is used to refer to the "linking of polymer chains" for both sciences, the extent of crosslinking and specificities of the crosslinking agents vary greatly. As with all science, there are overlaps, and the following delineations are a starting point to understanding the subtleties. Polymer chemistry Crosslinking is the general term for the process of ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
RNA Immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. Types Individual protein immunoprecipitation (IP) Involves using an antibody that is specific for a known protein to isolate that particular protein out of a solution containing many different proteins. These solutions will often be in the form of a crude lysate of a plant or animal tissue. Other sample types could be body fluids or other samples of biological origin. Protein complex immunoprecipitation (Co-IP) Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co- ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) 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 (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight. Properties SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. The polyacrylamide-gel is typically sandwiched between two glass plates in a slab gel. Although tube gels (in glass cylinders) were used historically, they were rapidly made obsolete with the invention of the more convenient slab gels. In addition, SDS (sodium dodecyl sulfate) is used. About 1.4 grams of S ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Unique Molecular Identifier
Unique molecular identifiers (UMIs), or molecular barcodes (MBC) are short sequences or molecular "tags" added to DNA fragments in some next generation sequencing library preparation protocols to identify the input DNA molecule. These tags are added before PCR amplification, and can be used to reduce errors and quantitative bias introduced by the amplification. Applications include variant calling in ctDNA, gene expression in single-cell RNA-seq (scRNA-seq) and haplotyping via linked reads. See also * Batch effect * Multiplex (assay) In the biological sciences, a multiplex assay is a type of immunoassay that uses magnetic beads to simultaneously measure multiple analytes in a single experiment. A multiplex assay is a derivative of an ELISA using beads for binding the capture a ... References DNA sequencing {{genetics-stub ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
PAR-CLIP
PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) is a biochemistry, biochemical method for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of ribonucleoside analogs that are photosensitivity, photoreactive, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG), into nascent RNA transcripts by living cells. Irradiation of the cells by ultraviolet light of 365 nm wavelength induces efficient cross-link, crosslinking of photoreactive nucleoside–flow tracer, labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a complementary DNA, cDNA library and is deep sequencing, deep sequenced using next-generation sequencing technology. Recently, PAR-CLIP have been applied to determine the ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
HITS-CLIP
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to generate genome-wide protein-RNA interaction maps for the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2; since then a number of other splicing factor maps have been generated, including those for PTB, RbFox2, SFRS1, hnRNP C, and even N6-Methyladenosine (m6A) mRNA modifications. HITS-CLIP of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets by decoding microRNA-mRNA and protein-RNA interaction maps in mouse brain, and subsequently in ''Caenorhabditis elegans'', embryonic stem cells and tissue culture cells. As a novel modification of HITS-CLIP, m6A-CLIP was developed to precisely map N6-Methyladenosine(m6A) locations in mRNA by UV-crosslinking m6A antibody to the targ ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Biochemistry Detection Methods
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and metabolism. Over the last decades of the 20th century, biochemistry has become successful at explaining living processes through these three disciplines. Almost all areas of the life sciences are being uncovered and developed through biochemical methodology and research. Voet (2005), p. 3. Biochemistry focuses on understanding the chemical basis which allows biological molecules to give rise to the processes that occur within living cells and between cells,Karp (2009), p. 2. in turn relating greatly to the understanding of tissues and organs, as well as organism structure and function.Miller (2012). p. 62. Biochemistry is closely related to molecular biology, which is the study of the molecular mechanisms of biological phenomena.Astb ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Genetics Techniques
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar working in the 19th century in Brno, was the first to study genetics scientifically. Mendel studied "trait inheritance", patterns in the way traits are handed down from parents to offspring over time. He observed that organisms (pea plants) inherit traits by way of discrete "units of inheritance". This term, still used today, is a somewhat ambiguous definition of what is referred to as a gene. Trait inheritance and molecular inheritance mechanisms of genes are still primary principles of genetics in the 21st century, but modern genetics has expanded to study the function and behavior of genes. Gene structure and function, variation, and distribution are studied within the context of the cell, the organism (e.g. dominance), and within the context o ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
