| 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. | View Page |
| Agarose Gel Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins.Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples. | View Page |
| Polyacrylamide Gels 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. | View Page |
| 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 |
| Routine Electrophoresis Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis performed on a rectangle-shaped slab gel. Routine electrophoresis is mostly used for separation of proteins and has some use in separating nucleic acids. Generally several patient specimens and control(s) can be placed on one gel and solutes separated in one run. This type of electrophoresis is sometimes called zone electrophoresis.A serum sample with normal plasma proteins yields five zones or bands of separated proteins: albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, and gamma-globulins. Proteins in CSF and urine proteins are also separated with routine electrophoresis. Using whole blood treated with a reagent to lyse red blood cells, variant and glycosylated hemoglobins can be detected. With different visualization methods, isoenzymes and lipoproteins in a serum sample can be identified.A manual agarose gel electrophoresis of eight serum samples is pictured below. After electrophoresis, the gel was stained with Ponceau S. | View Page |
| 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. | View Page |
| 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. | View Page |
| Two-Dimensional Electrophoresis 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. | View Page |
| Immunofixation Electrophoresis An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma.Below is the immunofixation electrophoresis gel from a serum sample analyzed on SPIFE 3000, Helena Laboratories. After electrophoresis, the precipitated proteins are stained with Acid Violet, a stain developed and used by Helena Laboratories. The SP lane represents a routine serum protein electrophoresis of this specimen. On the next three protein separations, antiserum against IgG, IgA, and IgM were applied to the G, A, M lanes respectively. Antiserum to kappa light chain was added to the next protein separation and antiserum to lambda light chain to the last protein separation. | View Page |