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| Chem 431 |
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Fall 2008 |
| Lecture Notes:: 6 November |
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In electrophoresis particles move in an electric field at rates depending on their size, shape, and electrical charge. Electrophoresis was first developed by Tiselius early in this century. Originally used moving boundary electrophoresis where the molecules in a disk of solution moved in bands from the center of a cell of solution towards electrodes at either end. Unfortunately diffusion, and more importantly, heat driven convection, caused serious band broadening, greatly reducing potential resolution.
Today nearly all electrophoresis is run as zone electrophoresis, meaning it is run in a support, such as a gel, with a small pore structure which essentially eliminates bulk convective flow and thus gives less band broadening and higher resolution. (An alternative high resolution strategy, which we will not study, is capillary electrophoresis. In this case the small diameter of the tube restricts convection and again high resolution can be achieved.)
The Basis of electrophoresis is movement in an electric field driven by an electrostatic force:
where E is the electric potential (volts), Q is the charge on the particle, and d is the distance over which the potential is applied (in effect the distance between the electrodes). This force will of course cause the particles to accelerate, but as the velocity increases an opposing , frictional force, will arise:
v
where v is the velocity of the particle and (eta) is the viscosity of the solution. (Notice that this equation
is based on the behavior of a sphere of radius = r in a viscous
solution - corrections will be needed for non-spherical particles.)
Very quickly a steady state will arise where:
v
= 
= the electrophoretic mobility.
Notice that v 
Q/r so the
velocity is related to both the charge and size of the molecule.
For non-spherical particles shape will also impact velocity since
our derivation was based on friction for a sphere.
The charge and apparent size of a particle can be affected by the type of solution:
A variety of support media have been used in electrophoresis, including paper, starch and a variety of gels. The most important for biochemistry are the polyacrylamide and agarose gel systems. Both of these systems allow a variation in pore size in the gel matrix, an important consideration when attempting to separate or characterize macromolecules by size. Agarose is particularly used for larger macromolecules such as DNA since it has greater strength at large pore sizes. Large pore size allows the Characterization of larger macromolecules, but poorly resolves differences in in size for smaller macromolecules. Larger pore size also generally results in a weaker gel matrix: that is the gels become more delicate and harder to handle.
Polyacrylamide is a synthetic, chemically polymerized gel widely used in biochemistry and molecular biology. The pore size can be readily manipulated by varying the concentration of monomer in the gel or the degree of cross-linking. Practically it is easier to manipulate pore size by varying concentration. Note that the pore size will vary statistically - varying monomer concentration varies the average pore size. Useful molecular weight ranges, Mr (the subscript r is used because they are actually based on Stokes radius since the gel is really separating by size, not molecular weight):
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PAGE can be used in either of the common electrophoretic modes: native or SDS/denaturing. In the native mode the mobility is affected by molecular weight, shape, and charge. It is thus not useful for characterizing molecules, though it can be very useful for separating mixtures of proteins.
Under reducing conditions (in the presence of sulfhydryl reagents) the denaturing detergent sodium dodecyl sulfate (SDS) will convert proteins into long rods with a uniform charge density. As a result, in an electric field, they will be subject to a uniform force field (gravitation would be analogous). Under these conditions, in an open solution all of the particles would migrate at the same rate. If they are now forced to migrate though a matrix of various hole sizes, as exist in the gel, the migration of larger molecules will be retarded relative to the smaller molecules. Importantly, the mobility is dependent on a single character. Note that if we now compare molecules of similar type (proteins for example) the sizes will be proportional to molecular weight (MW). Thus if we run an unknown against a set of known MW standards we can determine MW for the unknown with reasonable precision.
As shown in the figure at left, a plot of log MW vs relative mobility will give a straight line.
(Note that DNA can be treated similarly with PAGE except that SDS is unnecessary since DNA is already a "linear" molecule with uniform charge density from the backbone phosphates.)
Discontinuous electrophoresis is designed to sharpen the bands and increase the resolution of various electrophoretic methods. In the technique a "stacking gel" is layered on above the running or resolution gel. The stacking gel is very dilute so as to allow unimpeded passage of all of the proteins in a run. It is also set up with a different pH (generally differs from the running gel by about 2 units) and buffer system. The net result is that the proteins migrate very rapidly through the stacking gel, then "slam" into the running gel and stack up in a very thin band. The proteins are thus concentrated giving both better resolution and higher sensitivity.
In electrofocusing we take advantage of the fact the a molecule will not migrate in an electric field at its pI (the pH at which it has no net charge). Since proteins generally have many different charge side chains in many different local environments (affecting their pKas), each protein will have unique pI. The strategy in electrofocusing is then to set up a pH gradient in a media such as polyacrylamide gel, introduce a protein mixture, and then subject it to an electric field. Proteins will then migrate toward the positive or negative electrode until they reach a pH equivalent to their pI, at which time they cease migration. Note that the protein can initially be distributed throughout the gel since migration takes place in both directions!
One of the most powerful techniques in protein separation results from the combination of electrofocusing and SDS-PAGE in a 2-D system. Note that the electrofocusing and PAGE distinguish between proteins based on completely independent properties - pI and MW. In a 2-D system one first uses electrofocusing to separate the proteins (e.g. in a small tube or capillary electrophoresis system), then lays the electrofocusing gel across the top of a SDS-PAGE slab gel and runs the proteins down into the SDS-PAGE gel.
 Laboratory |
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Last modified 6 November 2008