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Electrophoretic Mobility and Electrophoresis (24.10). Electrical force is another way we can cause macromolecules to move Macromolecules tend to have charges associated with them when in solution (e.g., proteins)
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Electrophoretic Mobility and Electrophoresis (24.10) • Electrical force is another way we can cause macromolecules to move • Macromolecules tend to have charges associated with them when in solution (e.g., proteins) • Electrical force is proportional to the number of charges on the macromolecule, which is related to the size of the macromolecule • A steady-state motion of the molecule is achieved when the electrical and frictional forces balance each other out • Electrophoretic mobility (μ) is similar to sedimentation coefficient, but is not as easily obtained • Electrophoresis is the use of electrical force to separate and characterize proteins and nucleic acids • Different sized biomolecules migrate through the sample at different rates • Mass determinations are accomplished by comparing to a set of standards
Methods in Electrophoresis (24.10) • Gels are used to “slow down” biomolecule motion in order to achieve greater separation • Polyacrylamide or agarose gels increase frictional forces, so they lower μ • Gels act as molecular sieves, so they separate molecules by size • Nucleic acids are often separated based on size • As mass increases (more bp added), frictional forces increase but so does the number of charges (phosphates) • As size increases, molecular sieving of gels help to separate nucleic acids • Pulsed field electrophoresis can be used for very large nucleic acid structures, where structures get tangled in the gel (100-10000 kbp) • Proteins can be separated by size and charge • SDS electrophoresis operates in a similar fashion to gel electrophoresis of nucleic acids • Isoelectric focusing relies on a pH gradient in the gel to separate proteins • Protein motion stops when protein becomes neutral (isoelectric point or pI) • SDS electrophoresis and isoelectric focusing can be coupled together (2-D)
Elementary Chemical Kinetics (25.1-25.2) • Kinetics is the study of how reactions occur • Speed of reaction depends on frequency of productive collisions between molecules (concentration, temperature, nature of productive collision) • Many reactions involve more than one step, so a mechanism is used to explain how the reaction occurs • Reaction rates are measured as the speed with which a reactant is consumed or a product is created • Reaction rate is a differential equation since we are looking at a change in concentration in a given amount of time • One typically monitors either decay of one reactant or the production of a single product • Accomplished through absorbance, fluorescence, pH, etc.
