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SDS PAGE - Term Paper Example

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First-name Last Number 9 April 2013 SDS PAGE Background The method of separation of macromolecules in an electric field is called electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is a method of separating protein molecules based on their ability to move through an electric field depending on size of their polypeptide chains and molecular weight…
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Download file to see previous pages -mecaptoethamol prevents oxidation of cysteines and break up disulphide bonds, Bromophenyl dye is used to help in visualizing the sample as it travels across the gel and glycerol is added to make the sample fall to the bottom. (Leamlli, 1970). Electric charge is applied across the gel to influence the movement of protein molecules to positively charged end. Principal behind SDS PAGE The structure of protein molecules to be separated has portions of negative and positive charges resulting from charged R-groups of particular amino acid and hydrophobic portions of nonpolar R-groups hence difference in shapes and sizes of protein molecules. (Hempelmann, 2008) Therefore, the first stage of SDS PAGE is to make the protein molecules linear and to ensure that there are no secondary, tertiary, or quaternary molecular structures in the sample. This is achieved by injecting the sample with SDS, which is a detergent that can dissolve the membrane and solubilise the protein molecules. Its negative charge overcome positive charges of protein molecules. The resulting protein is denatured, linearized, and negatively charged. The next stage in SDS PAGE is the separation process. This is based on the molecular size and weight of the molecules, sieving properties of the gel is of great assistance at this stage since protein molecules have the same charge-to-mass-ratio. The gel is not a solid but made up of series of tunnel of different diameters running from one end to the other and are scattered through the gel. (SDS PAGE, 2009) Velocity of particles moving through an electric field is directly proportional to strength of the electric field and degree of charge in the particle but inversely proportional to size of the particles and viscosity of the medium. This is the basis upon which protein of different sizes are separated. (SDS PAGE. 2009). The discontinuous pH parts of the gel come handy in aligning the proteins properly at the starting point. Laemmli gel is composed of stacker and running gel, the running gel is buffed with Tris to pH of 8.8 with HCl; stacker gel is adjusted with Tris to pH 6.8 with HCl. The electrode buffer is adjusted to pH 8.3 using glycine and the gel is then run at a constant voltage. (Leamlli, 1970) When the power is switched on, glycine (which is a weak acid and can exist in an uncharged state as zwitterions at low pH, or in a charged state as glycinate anion at high pH) ions in the running buffer wants to move away from the negative electrode towards the sample and the stacking gel. The pH in stacking gel is low and so glycin ions lose their charge and slow down. In the stacker and sample, negatively charged mobile chloride ions move away from the cathode creating a narrow zone of very low conductance (very high electrical resistance) in the top of the stacking gel. Almost all the applied voltage is concentrated in this small zone. The very high field strength makes the negatively charged proteins to move forward. The trick, however, is that they can never outrun the chloride ions. If they did, they would find themselves in a region of high conductance and very low field strength and would immediately slow down. The result is that all the proteins move through the stacker in a tight ...Download file to see next pagesRead More
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