Structural Biochemistry/Proteins/Gel Electrophoresis
Gel electrophoresis is a technique used to display and assert that the purification scheme was effective by measuring the number of different proteins in a mixture. The basis of gel electrophoresis is the fact that molecule with specific net charge will move through an electric field. The speed of protein migration can be quantified as:
With E as magnitude of the electric field, z as net charge of a protein, and f as frictional kinetic coefficient.
Frictional coefficient, for spherical molecule, is determined as:
f = 6 π η r
with η as viscosity.
As its equation implies, the velocity of molecule traveling in the gel matrix depends on its size, shape, and the charge that it has. The smaller the molecule, the faster it will travel. Furthermore, Gels can be made in a variety of wt percents: 6%, 8%, 10%, 12% and 15%. Higher percentages are used primarily for smaller molecules and smaller percentages are used for larger sized samples. Theoretically, larger molecules can still be used with higher percents, but these gels may take a long time to develop. Charge can also be a factor in the speed and distance that a specific sample travels through the gel. Using a higher voltage will send the samples farther and faster. However, caution must be used with higher voltages as the heat it generates may melt the gels.
Gel Electrophoresis (SDS-PAGE; SDS-polyacrylamide Gel Electrophoresis) is a powerful tool to check the purity of the sample because because it can detect minuscule amount of protein. Different proteins appear as different bands on SDS-Polyacrylamide Gel after gel has been stained with Coomassie blue (visualize ~2pm of protein) or silver stain (visualize 0.02 µg of protein).
Native Gel Electrophoresis
Native Gel Electrophoresis involves running gels with samples in its native state. In doing so, the charge of the molecule becomes a factor in addition to size. More specifically, more charged molecules will migrate faster and farther than less charged molecules of comparable mass. Likewise, larger molecules will migrate less and at slower speeds than another molecule of comparable charge. Native Gel Electrophoresis most often involves two types of gels - Agarose and Polyacrylamide. Agarose is a derivative of the cell membranes of red algae composed of polysaccharides agarose and agaropectin, and due to the larger size of the pores, agarose gels are better suited for protein samples larger than 200 kilodaltons. Polyacrylamide (poly 2-propenamide, is a readily-crosslinked polymer of the neurotoxin acrylamide. It's pores are more fine, and while agarose is most commonly used for most cases, polyacrylamide is the gel of choice for smaller sample masses.
The Use of SDS (sodium dodecyl sulfate)
Electrophoresis involves the movement of particles due to electric current on the basis of size, charge of molecules, amount of current, etc. SSDS PAGE is a technique used to separate proteins based on size, and size alone. Sodium dodecyl sulfate (SDS) is a detergent that binds to proteins at every 2 amino acids in its sequence, and as SDS is very negative on its own, it changes the overall charge of the molecule to a negative charge. This negative charge is proportional to the protein's mass on the basis that the amount of SDS bound to the molecule is based on how many doublets of amino acids are present. The negative charge put on the protein is much larger than the charge originally there, which allows for a similar charge-to-mass ratio between different proteins. When SDS binds to proteins, it also changes the conformation of the proteins into similar shapes by denaturing the proteins and changing its bonds. SDS allows gel electrophoresis to separate proteins based on their molecular weights since the mass-to-charge ratio is relatively uniform among the proteins. This is because the SDS gel has sieving properties (offers resistance to particles based on their size)and is a uniform environment. It increases the differential mobility. The mobility of these proteins are then linearly proportional to the logarithm of their mass. Using this information, we can conclude from their mobility the mass of the protein and can even distinguish proteins that have a 2% difference in mass. Thus, the largest molecules, the ones that have more SDS bound to them, will fall down the electric field slower than the ones that have a smaller mass, and less SDS bound to them. This principle is contrary to the one in size-exclusion (gel-filtration) chromatography, which causes heavier molecules to come down first while the lighter ones come out later.
Certain solvents, such as PEG, glycerol, ethanol, and isopropanol, have an effect of decreasing the hydrodynamic radius of the proteins by decreasing the amount of free water to provide hydration spheres for the proteins. The polar solvents will hydrogen bond with the water, decreasing the disorder around the proteins and as a result, reducing the size of the hydration sphere. In such case, proteins will be eluted at a later stage as if they were of smaller size.
After the process is complete, the proteins are stained with a dye, forming bands, which represent the layers of mobility of each protein. With each additional purification process, the electrophoresis yields less bands, but a single darker band, which consequently represents the increased presence of the protein being isolated.
Two-Dimensional Gel Electrophoresis
The separation techniques of SDS-PAGE and isoelectric focusing can be utilized in conjunction to allow for 2DGE, which employs higher resolution and sensitivity in the separation of proteins. The first dimension of this powerful technique is isoelectric focusing (IEF) and the second dimension is polyacrylamide gel electrophoresis (PAGE). In the first dimension, proteins are separated according to their isoelectric point (pI). To do so, the gel is applied to the top of an SDS-polyacrylamide slab. Electrophoresis is then applied horizontally across the top of the gel and the proteins migrate into the second-dimension gel. Electrophoresis will then be applied again, this time vertically across the gel slab, and the proteins will migrate based on their molecular size. Heavier proteins will move shorter distances. Conversely, lighter proteins will move further.
While Two-Dimensional Gel Electrophoresis is a powerful technique that presents a higher resolution of separation, it does have its own limitations. 2DGE is a time-consuming and labor-intensive process, requiring manual gel polymerization, staining, and hours upon hours of separation. Furthermore, the technique is not without risk. Because heating of the gel may cause warping and diffusion of the molecules on the gel surface, 2DGE is difficult to reproduce.
Gel Electrophoresis in DNA Fingerprinting
DNA fingerprinting is a technique used to differentiate between different organisms based on the differences between each organism’s DNA configuration. DNA fingerprinting is often used by forensics labs to identify criminals by comparing a suspect’s DNA to the DNA found at a crime scene. DNA from the suspect is run through a gel electrophoresis and compared to a sample of DNA that was found at the scene. If the two samples produce identical band patterns in the gel, then confirmation that the suspect was at the scene of the crime can be made, since no two people possess identical patterns in their DNA.
In order to perform a fingerprint, a sample containing DNA must be obtained from each organism under evaluation. Examples of DNA samples include blood, urine, saliva, skin or hair. Before the samples can be analyzed, they must first be prepared. Preparation includes using restriction enzymes to separate the DNA into smaller pieces. Restriction enzymes are enzymes that cut DNA strands at specific nucleotides. These nucleotides are called restriction sites, and typically mark the end of a 4-8 unit sequence in nucleotides. The components and length of each restriction sequence vary from person to person, thus the use of restriction enzymes is an efficient way of separating an organism’s DNA into unique and specific sections. Additionally, certain amount of chemicals are also inserted as dye into the gel which will illuminate under UV light. This causes the bands to be much more visible when analyzing the sample protein.
Regions of DNA that contain many different short repeated sequences are called microsatellites. The lengths of these microsatellites vary greatly from person to person, which makes them prime locations for restriction enzymes to fragment the DNA. After treating the DNA samples with restriction enzymes, the DNA is now ready to be analyzed. The samples are loaded into the wells in a slab of gel, and an electric current is applied. Smaller fragments of DNA run through the gel faster, and will therefore be closer to the bottom, while larger fractions remain closer to the top. If two samples of DNA are run at the same time, the locations of the bands can be compared. If the patterns of bands between the two samples are identical, it means that the restriction enzymes partitioned each sample’s DNA at the same locations, indicating the two DNA samples had identical nucleotide sequencing. Identical nucleotide sequencing reveals the two samples are from the same organism.
DNA fingerprinting is also a useful technique to determine whether or not two people are related. Although no two people share the same DNA patterns, sections of microsatellites are passed down from parent to child. Not all of these sections are passed down, but offspring do not contain any pattern that their parents did not possess. A paternity or maternity test can be performed by comparing the DNA fingerprint of the individuals in question. If there are large groups of patterns that repeat in each sample’s fingerprint, it is likely that the individuals are related. The embedded image contains a three different DNA fingerprints, as indicated by the three different patterns of bands. Although these patterns represent fingerprints from different people, sample 2 shares similar patterns with both 1 and 3, which indicates that the person whose DNA is represented by sample 2 is likely to be the child of sample 1 and 3.
Maternal and paternal DNA fingerprinting tests are used to determine the probability of two people being related. These tests do not give definitive answers, and are not foolproof.
Visualization of protein in gels
As most proteins are not directly visible on gels to the naked eye, a method has to be employed in order to visualize them following electrophoresis. The most commonly used protein stain is the dye Coomassie brilliant blue. After electrophoresis, the gel containing the separated proteins is immersed in an acidic alcoholic solution of the dye. This denatures the proteins, fixes them in the gel so that they do not wash out, and allows the dye to bind to them. After washing away excesse dye, the proteins are visible as discrete blue bands. As little as 0.1-1.0 µg of a protein in a gel can be visualized using Coomassie brilliant blue. A more sensitive general protein stain involves soaking the gel in a silver salt solution. However, this technique is rather more difficult to apply. If the protein sample is radioactive the proteins can be visualized indirectly by overlaying the gel with a sheet of X-ray film. With time (hours to weeks depending on the radioactivity of the sample proteins), the radiation emitted will cause a darkening of the film. Upon development of the film the resulting autoradiograph will have darkened areas corresponding to the positions of the radiolabeled proteins. Another way of visualizing the protein of interest is to use an antibody against the protein in an immunoblot (Western blot). For this techinique, the proteins have to be transferred out of the gel on to a sheet of nitrocellular or nylon membrane. This is accomplished by overlaying the gel with the nitrocellulose then has an exact image of the pattern that was in the gel. The excess binding sites on the nitrocellulose are then blocked with a nonspecific protein solution such as milk powder, before placing the nitrocellulose in a solution cantaining the antibody that recognizes the protein of interest (the primary antibody). After removing excess unbound antibody, the primary antibody that is now specifically bound to the protein of interest is detected with either a radiolabeled, fluorescent or enzyme-coupled secondary antibody. Finally, the secondary antibody is detected either by placing the nitrocellulose against a sheet of X-ray film (if a radiolabeled secondary antibody has been used), by using a fluorescence detector or by adding to the nitrocellulose a solution of a substrate that is converted into a colored insoluble product by the enzyme that is coupled to the secondary antibody.
Hames, David. Hooper, Nigel. Biochemisty. Third edition. Taylor and Francis Group. New York. 2005.