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 (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 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 (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 productmonitors – Accomplished through absorbance, fluorescence, pH, etc.
Rate Laws and Reaction Mechanisms (25.3-25.4) 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