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Cycloheximide Chase
Cycloheximide chase assays are an experimental technique used in molecular and cellular biology to measure steady state protein stability. Cycloheximide is a drug that inhibits the elongation step in eukaryotic Translation (biology), protein translation, thereby preventing protein synthesis. The addition of cycloheximide to cultured cells followed by protein lysis at multiple timepoints is conducted to observe protein degradation over time and can be used to determine a protein's half-life. These assays are often followed by western blotting to assess protein abundance and can be analyzed using quantitative tools such as ImageJ. Implementation Cycloheximide chase assays have been conducted using a variety of cell types such as yeast and mammalian cell lines. Depending on the cell system used for analysis, the assay may vary in application and time course. For example, yeast cells expressing a protein substrate of interest typically require cycloheximide chases lasting up to 90 min ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metabolic reactions, DNA replication, Cell signaling, responding to stimuli, providing Cytoskeleton, structure to cells and Fibrous protein, organisms, and Intracellular transport, transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the Nucleic acid sequence, nucleotide sequence of their genes, and which usually results in protein folding into a specific Protein structure, 3D structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called pep ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Cycloheximide
Cycloheximide is a naturally occurring fungicide produced by the bacterium '' Streptomyces griseus''. Cycloheximide exerts its effects by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in relation to the ribosome), thus blocking eukaryotic translational elongation. Cycloheximide is widely used in biomedical research to inhibit protein synthesis in eukaryotic cells studied ''in vitro'' (''i.e.'' outside of organisms). It is inexpensive and works rapidly. Its effects are rapidly reversed by simply removing it from the culture medium. Due to significant toxic side effects, including DNA damage, teratogenesis, and other reproductive effects (including birth defects and toxicity to sperm), cycloheximide is generally used only in ''in vitro'' research applications, and is not suitable for human use as a therapeutic compound. Although it has been used as a fungicide in agricultural applications, this application is now decreas ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Translation (biology)
In biology, translation is the process in living Cell (biology), cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in the addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression. In translation, messenger RNA (mRNA) is decoded in a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later protein folding, folds into an Activation energy, active protein and performs its functions in the cell. The polypeptide can also start folding during protein synthesis. The ribosome facilitates decoding ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Lysis
Lysis ( ; from Greek 'loosening') is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles. Many species of bacteria are subject to lysis by the enzyme lysozyme, found in animal saliva, egg white, and other secretions. Phage lytic enzymes (lysins) produced during bacteriophage infection are responsible for the ability of these viruses to lyse bacterial cells. Penicillin and related β-lactam antibiotics cause the death of bacteria through enzyme-mediated lysis that occurs after the drug causes the bacterium to form a defective cell wall. If the cell wall is completely lost and ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Western Blotting
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 antibo ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitute 1% of all described fungal species. Some yeast species have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae, or quickly evolve into a Multicellular organism, multicellular cluster with specialised Organelle, cell organelles function. Yeast sizes vary greatly, depending on species and environment, typically measuring 3–4 micrometre, μm in diameter, although some yeasts can grow to 40 μm in size. Most yeasts reproduce asexual reproduction, asexually by mitosis, and many do so by the asymmetric division process known as budding. With their single-celled growth habit, yeasts can be contrasted with Mold (fungus), molds, wh ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
MG132
MG132 is a potent, reversible, and cell-permeable proteasome inhibitor ( Ki = 4 nM). It belongs to the class of synthetic peptide aldehydes. It reduces the degradation of ubiquitin-conjugated proteins in mammalian cells and permeable strains of yeast by the 26S complex without affecting its ATPase or isopeptidase activities. MG132 activates c-Jun N-terminal kinase (JNK1), which initiates apoptosis. MG132 also inhibits NF-κB activation with an IC50 of 3 μM and prevents β-secretase cleavage. Molecular mechanism There are several inhibitors that can readily enter cell and selectively inhibit degradative pathway. It includes peptide aldehydes, such as Cbz-leu-leu-leucinal (MG132), Cbz-leu-leu-norvalinal ( MG115) and acetyl-leu-leu-norleucinal ( ALLN). These are substrate analogues and potent transition-state inhibitors of chymotrypsin like activity of proteasome machinery. The peptide aldehydes are also known to inhibit certain lysosomal cysteine proteases and the calpai ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
