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Biophysical Chemistry

Biophysical Chemistry. Gel Electrophoresis. Definition. Electro = Charge + Phorsesis= Carry Electrophoresis = Separation of charged molecules by differences in their rate of migration in an electric field. The Components of The System. Molecules to be separated Proteins Nucleic Acids

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Biophysical Chemistry

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  1. Biophysical Chemistry Gel Electrophoresis

  2. Definition • Electro = Charge + Phorsesis= Carry • Electrophoresis = Separation of charged molecules by differences in their rate of migration in an electric field.

  3. The Components of The System • Molecules to be separated • Proteins • Nucleic Acids • Support medium • Gel (Starch, Polyacrylamide or Agarose) • Buffer System • High Buffer Capacity • DC Power Source • 50 – 1000 V

  4. Common Support Media for Biological Molecules • Proteins • Native Gel (Acrylamide or Starch) • Denaturing (SDS) Gel (Acrylamide) • Nucleic Acids – DNA & RNA • Agarose Gel • Acrylamide Gel

  5. Factors that Influence Mobility • Properties of the Molecules to be separated • Molecular size (MW) • Molecular shape • Molecular charge • Properties of the System • Electric field strength (V/cm) • Porosity of the support medium (% S) • Conductivity of the buffer (R) • pH of the buffer

  6. These Factors Interact • Mobility is proportional to charge/MW. • Charge is affected by buffer Ph • Mobility is proportional to field strength. • Allowable field strength is affected by buffer conductivity (high conductivity high current heat) • Mobility is inversely proportional to buffer conductivity.

  7. Making a Separation • Electrophoresis systems are designed to optimize the separation of specific molecule types based on specfic molecular parameters: • Nucleic acids: Charge/BP is a constant. Separation can be based on number of base pairs (given all molecules have same shape). Larger molecules move slower due to friction with gel • Proteins: Charge varies as a function of amino acid composition and buffer pH. Separation is based on Charge/MW (shape may also vary). The exact combination of factors varies for each molecule

  8. Separating Proteins Based on MW • The problem associated with protein separation (too many separation parameters) has been solved by using denaturing SDS Gels • The proteins are heat denatured which makes them all the same shape (linear) • The proteins are coated with an ionic detergent (SDS) which gives all molecules approximately the same overall negative charge • Separation is based on MW alone

  9. SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

  10. Separation of DNA Molecules in Agarose Gels • In most cases the molecules are linear • The phosphate groups bear negative charge at neutral pH (2 phosphates/BP) • Therefore mobility will be based on number of base pairs/molecule • The Procedure for making and running agarose gels is shown in the following video

  11. Physics Concepts Addressed • Basic DC circuits and Ohm’s law. • Ionic strength and conductivity • Ohmic heating • The concept of an electrical field. • Force acting on a charged molecule as a result of an applied voltage • Frictional resistance to mobility • Friction as a function of molecular surface area • The use of logarithmic paper (or spread sheets). • Determination of molecular size

  12. Practical Considerations for Teaching Gel Electrophoresis in the High School • Availability of materials and equipment • Science Kit & Carolina both have kits for doing DNA electrophoresis. • Cost (Demonstration vs. Hands on) • Safety issues • Toxic chemicals – minimal toxicity except for ethidium bromide stain. Can not be used!!! • Electrical hazards- 80 V DC is the standard voltage used in most setups. The power supplies in the kits are often limited to 100 V. GFI outlets are manditory.

  13. References and Web Sites • Polyacrylamide Gel Electrophoresis of Proteins and DNA-Modern Bio. Inc http://www.modernbio.com/polyacrimide.htm • Simple Electrophoresis of dyes http://gslc.genetics.utah.edu/units/activities/electrophoresis/ • Vivoy et al. The physics of DNA Electrophoresis. Contemp Phys. 33 1-40 (1992) • Physics and gel electrophoresis: using terminal velocity to characterize molecular weight http://www.iop.org/EJ/abstract/0143-0807/19/6/011 • Edvoteck http://www.southernscientific.com/apbiology_electrophoresis.asp • Wards http://wardsci.com/search.asp?t=ss&ss=Electrophoresis&x=14&y=8 • Nat Cent Biotech Edu. http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/DNA/baseunit.html • Physics at Work (Biophysics) http://www.phy.cam.ac.uk/camphy/outreach/physics_at_work_2003/exhibitor/biophysics.htm • Low toxicity stains http://www.bioscience-explained.org/EN1.2/schollar.html

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