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8.3: Electrophoresis

[ "article:topic", "authorname:ahern2", "Isoelectric focusing", "electrophoresis", "Polyacrylamide gel electrophoresis", "license:ccbyncsa" ]
  • Page ID
    10441
  • Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix, itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-moving.

    For DNA and RNA, sorting molecules by size in this way is trivial, because of the uniform negative charge on the phosphate backbone. For proteins, which vary in their charges, a clever trick must be employed to make them mimic nucleic acids - see polyacrylamide gel electrophoresis (PAGE) below. Different kinds of gels have different pore sizes. Like sieves with finer or coarser meshes, some gels do a better job of separating smaller molecules while others work better for larger ones. Gel electrophoresis may be used as a preparative technique (that is, when purifying proteins or nucleic acids), but most often it is used as an analytical tool.

    Agarose Gel Electrophoresis

    Agarose gel electrophoresis is a technique used to separate nucleic acids primarily by size. Agarose is a polysaccharide obtained from seaweeds (Figure 8.11). It can be dissolved in boiling buffer and poured into a tray, where it sets up as it cools (Figure 8.12) to form a slab. Agarose gels are poured with a comb in place to make wells into which DNA or RNA samples are placed after the gel has solidified. The gel is immersed in a buffer and a current is applied across the slab. Double-stranded DNA has a uniform negative charge that is independent of the sequence composition of the molecule. Therefore, if DNA fragments are placed in an electric field they will migrate from the cathode (-) towards the anode (+). The rate of migration is directly dependent on the ability of each DNA molecule to worm or wiggle its way through the sieving gel. The agarose matrix provides openings for macromolecules to move through. The largest macromolecules have the most difficult time navigating through the gel, whereas the smallest macromolecules slip through it the fastest.


    Figure 8.11 - Structure of the agarose polysaccharide. Wikipedia

    Because electrophoresis uses an electric current as a force to drive the molecules through the matrix, the molecules being separated must be charged. Since the size to charge ratio for DNA and RNA is constant for all sizes of these nucleic acids, the molecules simply sort on the basis of their size - the smallest move fastest and the largest move slowest.

    All fragments of a given size will migrate the same distance on the gel, forming the so-called “bands” on the gel. Visualization of the DNA fragments in the gel is made possible by addition of a dye, such as ethidium bromide, which intercalates between the bases and fluoresces when viewed under ultraviolet light (Figure 8.13) By running reference DNAs of known sizes alongside the samples, it is possible to determine the sizes of the DNA fragments in the sample. It is useful to note that, by convention, DNA fragments are not described by their molecular weights (unlike proteins), but by their length in base-pairs( bp) or kilobases (kb).

     

    Figure 8.12 - Agarose gel electrophoresis separation of DNA - orange bands are DNA fragments. Wikipedia


    Figure 8.13 - DNA bands visualized with ethidium bromide staining. Wikipedia

    Polyacrylamide gel electrophoresis (PAGE)

    Like DNA and RNA, proteins are large macromolecules, but unlike nucleic acids, proteins are not necessarily negatively charged. The charge on each protein depends on its unique amino acid sequence. Thus, the proteins in a mixture will not necessarily all move towards the anode.

    Additionally, whereas double-stranded DNA is rod-shaped, most proteins are globular (folded). Further, proteins are considerably smaller than nucleic acids, so the openings of the matrix of the agarose gel are simply too large to effectively provide separation. Consequently, unaltered (native) proteins are not very good prospects for electrophoresis on agarose gels. To separate proteins by mass using electrophoresis, one must make several modifications.

    Gel matrix

    First, a matrix made by polymerizing and cross-linking acrylamide units is employed. A monomeric acrylamide (Figure 8.14) is polymerized and the polymers are cross-linked using N,N’-Methylene-bisacrylamide (Figure 8.15) to create a mesh-like structure. One can adjust the size of the openings of the matrix/mesh readily by changing the percentage of acrylamide in the reaction. Higher percentages of acrylamide give smaller openings and are more effective for separating smaller molecules, whereas lower percentages of acrylamide are used when resolving mixtures of larger molecules. (Note: polyacrylamide gels are also used to separate small nucleic acid fragments, with some acrylamide gels capable of separating pieces of DNA that differ in length by just one nucleotide.)

    Figure 8.14 - Acrylamide monomer. Wikipedia

     

     


    Figure 8.15 - N,N’-Methylenebisacrylamide - acrylamide crosslinking reagent. Wikipedia

    Charge alteration by SDS

    A second consideration is that proteins must be physically altered to “present” themselves to the matrix like the negatively charged rods of DNA. This is accomplished by treating the proteins with the anionic detergent, SDS (sodium dodecyl sulfate). SDS denatures the proteins so they assume a rod-like shape and the SDS molecules coat the proteins such that the exterior surface is loaded with negative charges, masking the original charges on the proteins and making the charge on the proteins more proportional to their mass, like the backbone of DNA.

    Since proteins typically have disulfide bonds that prevent them from completely unfolding in detergent, samples are boiled with mercaptoethanol to break the disulfide bonds and ensure the proteins are as rod-like as possible in the SDS. Reagents like mercaptoethanol (and also dithiothreitol) are sulfhydryl-containing reagents that become oxidized as they reduce disulfide bonds in other molecules (see Figure 8.16)

    Figure 8.16 - Reduction of disulfide bonds by dithiothreitol. Wikipedia

    Stacking Gel

    A third consideration is that a “stacking gel” may be employed at the top of a polyacrylamide gel to provide a way of compressing the samples into a tight band before they enter the main polyacrylamide gel (called the resolving gel). Just like DNA fragments in agarose gel electrophoresis get sorted on the basis of size (largest move slowest and smallest move fastest), the proteins migrate through the gel matrix at velocities inversely related to their size. Upon completion of the electrophoresis, proteins may be visualized by staining with compounds that bind to proteins, like Coomassie Brilliant Blue (Figure 8.17) or silver nitrate.

    Figure 8.17 - Two SDS-PAGE gels - Proteins are the blue bands (stained with Coomassie Blue). Wikipedia
     

    Non-denaturing gel electrophoresis

    The SDS_PAGE technique described above is the commonest method used for electrophoretic separation of proteins. In some situations, however, proteins may be resolved on so-called “native” gels, in the absence of SDS. Under these conditions, the movement of proteins through the gel will be affected not simply by their mass, but by their charge at the pH of the gel, as well. Proteins complexed with other molecules may move as single entity, allowing the isolation of the binding partners of proteins of interest.

    Isoelectric focusing

    Proteins vary considerably in their charges and, consequently, in their pI values (pH at which their charge is zero). This can be exploited to separate proteins in a mixture. Separating proteins by isoelectric focusing requires establishment of a pH gradient in a tube containing an acrylamide gel matrix. The pore size of the gel is adjusted to be large, to reduce the effect of sieving based on size. Molecules to be separated are applied to the gel containing the pH gradient and an electric field is applied. Under these conditions, proteins will move according to their charge.

    Positively charged molecules, for example, move towards the negative electrode, but since they are traveling through a pH gradient, as they pass through it, they reach a region where their charge is zero and, at that point, they stop moving. They are at that point attracted to neither the positive nor the negative electrode and are thus “focused” at their pI (Figure 8.18). Using isoelectric focusing, it is possible to separate proteins whose pI values differ by as little as 0.01 units.

    Figure 8.18 - Isoelectric focusing: A. At the start of the run; B. at the end of the run

    2D gel electrophoresis

    Both SDS-PAGE and isoelectric focusing are powerful techniques, but a clever combination of the two is a powerful tool of proteomics - the science of studying all of the proteins of a cell/tissue simultaneously. In 2-D gel electrophoresis, a lysate is first prepared from the cells of interest. The proteins in the lysate are separated first by their pI, through isoelectric focusing and then by size by SDS-PAGE.

    Figure 8.19 - Scheme for performing 2-D gel analysis. Image by Aleia Kim

    The mixture of proteins is first applied to a tube or strip (Figure 8.19, Step 1) where isoelectric focusing is performed to separate the proteins by their pI values (Step 2). Next, as shown in the figure, the gel containing the proteins separated by their pIs is turned on its side and applied along the top of a polyacrylamide slab for SDS-PAGE to separate on the basis of size (Step 3). The proteins in the isoelectric focusing matrix are electrophoresed into the polyacrylamide gel and separated on the basis of size. The product of this analysis is a 2-D gel as shown in Figure 8.20.The power of 2-D gel electrophoresis is that virtually every protein in a cell can be separated and appear on the gel as a spot defined by its unique size and pI. In the figure, spots in the upper left correspond to large positively charged proteins, whereas those in the lower right are small negatively charged ones. Every spot on a 2-D gel can be eluted and identified by using high throughput mass spectrometry. This is particularly powerful when one compares protein profiles between different tissues or between control and treated samples of the same tissue.

     


    Figure 8.20 - Result of 2-D gel electrophoresis separation. Wikipedia

    Protein profiles comparison

    Comparison of 2-D gels of proteins from non-cancerous tissue and proteins from a cancerous tissue of the same type provides a quick identification of proteins whose level of expression differs between the two. Information such as this can be useful in designing treatments or in understanding the mechanism(s) by which the cancer develops.