Presentation on theme: "Electrophoretic Mobility and Electrophoresis (24.10) Electrical force is another way we can cause macromolecules to move – Macromolecules tend to have."— Presentation transcript:
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 Electrophoresis – 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 ( 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 Isoelectric focusing – 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 ( ) 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 productmonitors – Accomplished through absorbance, fluorescence, pH, etc.
Rate Laws and Reaction Mechanisms ( ) We know from experience that reaction rates often depend on concentration of reactants – Rate can be expressed as a product of reactant concentrations of certain orders – Order for each reactant is not necessarily the stoichiometric coefficient (α ≠ a) – Rate constant (k) must contain information about temperature and productive collisions Overall order of the reaction is the sum of the orders for each reactant and must be determined experimentally – The rate can be determined by measuring the change in concentration of a reactant/product over a short range of time (tangent to curve is rate)rate – The order for each reactant can be obtained by changing the concentrations of a single species and monitoring the change in rate (isolation method, method of initial rates) Reaction mechanism is a set of elementary reactions that can be used to explain a rate law – Order of reactants in an elementary rate law is the stoichiometric coefficient – Mechanism is only viable if the sum of elementary rate laws match overall rate law
Electrophoresis and Molecular Mobilities
Protein Electrophoretic Mobility and pH
Concentrations of Product and Reactant During Reaction