|Types of Electrophoresis|
There are numerous applications of electrophoresis. Routine protein electrophoresis performed in clinical laboratories is the oldest method and therefore the most frequently used method. With the advent of molecular diagnostics, several other electrophoresis methods have become very important, highly automated, and have several important applications.Types of electrophoresis that will be discussed are:Routine electrophoresisHigh resolution electrophoresisPolyacrylamide gel electrophoresisCapillary electrophoresisIsoelectric focusingImmunochemical electrophoresisTwo-dimensional electrophoresisPulsed field electrophoresis
|Types of Support Media|
For electrophoretic separation of solutes, the sample of solutes is placed on a gel or membrane in contact with buffer for separation. Common gels are cellulose acetate, agarose, and polyacrylamide gels. These gels are formed into sheets, slabs, or inserted into columns or tubes. The gel can be positioned horizontally or vertically.Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel. Because cellulose requires soaking before sample application and a clearing step for detection of separated solutes or bands, agarose gel is more often used than cellulose acetate gel for clinical electrophoresis.
Polyacrylamide electrophoresis (PAGE) is performed on a gel formed by polymerizing and cross-linking acrylamides. These gels are stronger than agarose gels and also thermostable and transparent. The matrix created by cross-linking the polymer chains is more regular and the pore sizes are more uniform in an individual gel. The pore size can be changed by changing the concentrations of the acrylamides used.In addition to separating fragments by charge and mass, PAGE also separates solutes by molecular size. When using PAGE, the gel allows more fractions of smaller size to be detected than the traditional agarose gel methods.Care is required in polyacrylamide gel preparation and use because acrylamides are carcinogenic.
|There are several different types of media that can be used in electrophoresis. Most methods today use a gel, cellulose acetate, agarose, or polyacrylamide gel. Which one of the following statements is true regarding these gels?||View Page|
|Of the three types of gels discussed, agarose gels are stronger, thermostable, and transparent.||View Page|
|Polyacrylamide Electrophoresis (PAGE)|
More separations are also achieved with layers of polyacrylamide gels each with a different pore size. The gels can be horizontal or vertical slabs or incorporated into vertical cylinders or rods. Varying the pore size in each layer is significant especially if very small pore sizes are created. DNA of 100 base pairs (bp) or less can be separated.Common applications of PAGE are separation of proteins and nucleic acids. Polyacrylamide gels are also used as the medium in several other types of electrophoresis described in this section.
|Denaturing Polyacrylamide Gels|
Denaturing chemicals can be added to the acrylamides during formation of polyacrylamide gels. These additives keep the solutes or molecules in a denatured state during separation. Urea denatures double-stranded DNA to single-stranded DNA. A detergent, sodium dodecyl sulfate (SDS), denatures proteins. Adding SDS with heat denatures proteins to small, similar shaped particles and coats each so that protein structures are not reformed. SDS is usually added to the gel and the protein sample. Then the mixture of protein coated fragments moves through polyacrylamide gel pores with speed similar to a mixture of DNA fragments.
|Isoelectric Focusing (IEF)|
Isoelectric focusing is a type of separation where the solutes migrate based upon a different principle. The separation takes place on a gel where a pH gradient has been created using ampholytes. Ampholytes are a mixtures of amphoteric polyaminocarboxylic acids. This mixture possesses a range of pIs, a high buffering capacity at each pH, and is used to create pH gradients.When ampholytes undergo electropohoresis, each individual ampholyte migrates to its own region, an area that matches its pI. After migration of ampholytes, the gel has stable pH zones of increasing pH or a pH gradient. The solutes in the specimen do not migrate to the electrode of opposite charge but to the zone or area that matches their pI. IEF is performed on a gel in a capillary tube, strip, or plate. Gels used are most commonly polyacrylamide gels but agarose and cellulose acetate can also be used.
|Capillary Electrophoresis (CE)|
Capillary electrophoresis (CE) combines electrophoresis and high performance liquid chromatography. CE takes place in a very thin, fused silica capillary tube with polyacrylamide or agarose gel. Polyacrylamide is the most common gel used. The ends of the capillary tube are placed in two buffer reservoirs with the anode in one, and the cathode in the other. A high voltage power supply and cooling system are included.One major difference in CE is the detection of separated solutes as migration and separation occur, instead of detection after separation. An optical detector attached to the capillary detects solutes after separation but while still in the capillary; the detector is linked to data collection and storage.
Two-dimensional electrophresis is separating the same sample with two distinct separation techniques or two different electrophoresis separations. The separated bands from one electrophoresis are resolved more with the second electrophoresis. IEF followed by PAGE or AGE is the most frequent two-dimensional electrophoresis. The gel from the IEF capillary is removed and placed across the PAGE or AGE gel slab at right angles for the second electrophoresis. If PAGE is used for the second electrophoresis, it is often PAGE with SDS.Two-dimensional electrophoresis can also be a single sample run on either agarose or polyacrylamide gels. The gel is then turned 90 degrees and the same type electrophoresis is run on the separated solutes to separate each band from the first run into more bands.The image below shows a two-dimensional electrophoresis separation of proteins which is IEF followed by PAGE with SDS. The proteins were first separated by IEF on a very narrow gel strip. This strip was then positioned at top of a polyacrylamide gel with SDS for the second electrophoresis. The IEF gel is the very narrow strip on top and remainder of the image is the many separated proteins on the PAGE with SDS.
|Currently there has been a revitalization in the clinical usage of electrophoresis. Previously, methods were primarily used to separate proteins in blood and other body fluids. From the following statements, select the statements that correctly describe newer applications of electrophoresis.||View Page|
|Sodium dodecyl sulfate is added to polyacrylamide gels to denature the proteins in the sample and enhance their separation.||View Page|
Successful electrophoresis requires application of correct amount of sample with clean applicators and no air bubbles. Many gel electrophoresis for manual sample application include a sample application template to lay over the gel, a micropipet, or a thin-wire applicator. Polyacrylamide gels will have wells in the gel for the sample. Sterile pipet tips are required when applying DNA and RNA specimens.