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7.25D: Western Bolts - Biology

7.25D: Western Bolts - Biology


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Learning Objectives

  • Show the uses of Western Blots

The Western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Western blot samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using either a blender (for larger sample volumes), a homogenizer (smaller volumes), or by sonication. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. The technique uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.

The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins belonging to signaling pathways or cell surface receptor antigens, or other cellular or soluble components. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines. Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA). This method originated in the laboratory of George Stark at Stanford. The name Western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed Northern blot.

Key Points

  • After separation by gel electrophoresis using SDS-PAGE, proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.
  • The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody is conjugated to alkaline phosphatase or horseradish peroxidase.
  • The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

Key Terms

  • electrophoresis: a method for the separation and analysis of large molecules (such as proteins) by migrating a colloidal solution of them through a gel; gel electrophoresis

Polyacrylamide gel electrophoresis

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. [1]

Hydration of acrylonitrile results in formation of acrylamide molecules ( C
3 H
5 NO ) by nitrile hydratase. [2] Acrylamide monomer is in a powder state before addition of water. Acrylamide is toxic to the human nervous system, therefore all safety measures must be followed when working with it. Acrylamide is soluble in water and upon addition of free-radical initiators it polymerizes resulting in formation of polyacrylamide. [2] It is useful to make polyacrylamide gel via acrylmide hydration because pore size can be regulated. Increased concentrations of acrylamide result in decreased pore size after polymerization. Polyacrylamide gel with small pores helps to examine smaller molecules better since the small molecules can enter the pores and travel through the gel while large molecules get trapped at the pore openings.

As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure. This method is called native-PAGE. Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length (because the protein-SDS complexes all have a similar mass-to-charge ratio). This procedure is called SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids. [3] : 164–79 In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains. When subjected to an electric field in PAGE, the negatively charged polypeptide chains travel toward the anode with different mobility. Their mobility, or the distance traveled by molecules, is inversely proportional to the logarithm of their molecular weight. [4] By comparing the relative ratio of the distance traveled by each protein to the length of the gel (Rf) one can make conclusions about the relative molecular weight of the proteins, where the length of the gel is determined by the distance traveled by a small molecule like a tracking dye. [5]

For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to coat proteins in order to impart two negative charges (from every SDS molecule) to every two amino acids of the denatured protein. [3] : 161–3 2-Mercaptoethanol may also be used to disrupt the disulfide bonds found between the protein complexes, which helps further denature the protein. In most proteins, the binding of SDS to the polypeptide chains impart an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content – for instance, many membrane proteins, and those that interact with surfactants in their native environment – are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS. [6] Procedurally, using both Native and SDS-PAGE together can be used to purify and to separate the various subunits of the protein. Native-PAGE keeps the oligomeric form intact and will show a band on the gel that is representative of the level of activity. SDS-PAGE will denature and separate the oligomeric form into its monomers, showing bands that are representative of their molecular weights. These bands can be used to identify and assess the purity of the protein. [3] : 161–3


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