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 Support medium –Gel (Starch, Polyacrylamide or Agarose) Buffer System –High Buffer Capacity DC Power Source –50 – 1000 V

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

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

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.

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

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

SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

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

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

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.

References and Web Sites Polyacrylamide Gel Electrophoresis of Proteins and DNA-Modern Bio. Inc Simple Electrophoresis of dyes Vivoy et al. The physics of DNA Electrophoresis. Contemp Phys (1992) Physics and gel electrophoresis: using terminal velocity to characterize molecular weight Edvoteck Wards Nat Cent Biotech Edu. Physics at Work (Biophysics) r/biophysics.htm r/biophysics.htm Low toxicity stains