| Amphoteric An amphoteric molecule has the ability to be negatively or positively charged. Changing the pH using buffers will alter the charge and magnitude of the charge. A molecule with this amphoteric ability is sometimes referred to as an ampholyte or even by the older term, zwitterion.Proteins with their ionizable amino and carboxyl groups are amphoteric. Nucleic acids (deoxyribonucleic acid or DNA and ribonucleic acid or RNA) are charged and thus are amphoteric. | View Page |
| The pI of a protein is 9.2. This protein is placed in an electrical field where a buffer sets the pH at 10.0. Select the correct statement regarding the electrophoretic migration of this protein. | View Page |
| Mobility or Rate of Migration The mobility or rate of migration of ions in electrophoresis is dependent upon the following factors: Net charge of the molecules Size and shape of the molecules Support medium properties Strength of the electrical field Ionic strength of the buffer Temperature | View Page |
| Rate of Migration The net charge of a molecule is the most important factor affecting the mobility of that molecule. The greater the net charge, the greater the mobility or the more quickly the molecule migrates. The net charge of a particular compound depends upon the buffer and the resultant pH set by that buffer. The size and shape of a molecule also influence the rate of migration in that the larger the size, the slower the molecule will move in electrophoresis.The viscosity and the pore size in the support media or gels used for electrophoresis influence the rate of migration. Increased viscosity slows the migration and increasing pore size speeds up the migration.Increased heat increases the rate of migration. Increasing the strength of the electrical field by increasing voltage and increasing the temperature used for the electrophoresis both increase the mobility and rate of migration. When increasing these factors that affect mobility, caution is necessary. Each will lead to an increase in temperature that can possibly denature the sample and alter the characteristics of the support medium. The ionic strength of the buffer and its effect on mobility are more complicated. The ionic strength of the buffer affects the thickness of the ionic cloud, the rate of migration, and the sharpness of the separated solutes. In electrophoresis, a cloud of ions forms over the medium and is composed of buffer ions, sample ions and other nonbuffer ions. Increasing the buffer ionic strength increases the buffer ions in the cloud and slows the movement of solutes and creates sharper bands. However, this also increases heat production. | View Page |
| Which one of the following will slow down the migration of solutes in electrophoresis? | View Page |
| Role of Buffers The two important purposes of the buffer are to create the pH and to conduct the current. The buffer ions will carry the current during electrophoresis. The pH set by the buffer determines the net charge on the solutes. The pH ionizes these solutes and the resulting net charge determines which electrode the solutes migrate toward. Besides setting the pH, the buffer also maintains the pH throughout the electrophoresis of the sample. | View Page |
| Buffers and pH The isoelectric point of most proteins is between pH 4.0 and 7.5. In pH 8-9, proteins will take on a negative charge and migrate to the anode. Most protein electrophoresis is performed at pH 8.6.Buffers most commonly used are barbital or tris-boric acid-EDTA buffers. They fix the pH at 8.6, leading to sharper bands and good separations. | View Page |
| Proteins in a buffer setting the pH at 8.6 will become anions and move to the positively charged electrode. | View Page |
| 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 |
| Electrophoresis Equipment In addition to the specimen sample, support medium and buffer for electrophoresis, a power supply, positive and negative electrodes, chamber, and identification or detection method are needed.The power supply is a source of constant voltage or current that provides energy to the electrodes. This drives the movement of the ions in the medium and results in the movement and separation of the molecules or solutes in the specimen. Control of current or voltage comes with the power source in order to make adjustments.The chamber is divided into two sections or has two reservoirs for the buffer and one electrode is placed in each. The support medium is laid over the chamber in such a way that it connects the two reservoirs. A lid or cover is placed over the chamber during electrophoresis. | 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 |
| In isoelectric focusing, the basis of separation of solutes is different than the other types of electrophoresis. Which statement below correctly describes this feature of isoelectric focusing? | View Page |
| Technical Considerations and Electrophoresis Troubleshooting Topics For successful gel electrophoresis, care must be taken with each of the following: Sample application Buffers Support medium StainsElectroendosmosis and wick flow are technical considerations. When irregular, distorted, and atypical bands result, possible causes are investigated. | View Page |
| Buffers To prevent microbial growth and contamination, buffers must be refrigerated when not in use. Using a buffer at refrigerator temperature also improves band resolution and lessens evaporation during electrophoresis. It is recommended that an electrophoresis using a small volume of buffer use a fresh buffer for each run. After a large volume buffer electrophoresis, the buffer can be reused if the buffer from each reservoir is combined, mixed well, and refrigerated. | View Page |
| Electroendosmosis With a pH 8.0-9.0 used for protein electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms. As the negative ion cloud migrates to the anode, the proteins are pulled to the anode. Several gels used routinely for protein electrophoresis attract positive ions from the buffer and form a positive ion cloud. This ion cloud moves in the opposite direction to the cathode. This phenomenon is called electroendosmosis or endosmosis.The tension created by these oppositely moving ion clouds can affect the movement of sample macromolecules. The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode. Many protein electrophoresis methods take advantage of this tension and use it to achieve better separation of protein bands. The gamma globulin band in serum, urine, and other body fluids will separate more sharply by being pushed to the cathode and will appear behind the point of sample application. | View Page |
| Wick Flow Wick flow is caused by the movement of buffer into the support medium. Moisture evaporation from the gel from heat generation during electrophoresis causes movement of the buffer into the gel. Gel absorption of buffer to replace the lost moisture affects the migration of sample molecules. Using a lid or cover during electrophoresis can prevent some of this evaporation. Methods that generate excessive heat utilize a cooling system during electrophoresis to prevent wick flow and other damage to sample solutes. | View Page |
| Troubleshooting Irregular, Distorted or Atypical Bands Band Appearance Possible Cause Short Migration Patterns Contaminated or Aged Buffer Diffuse Bands Markedly Wet Gels Poor Sample Application Streaks Perpendicular to Bands Tearing/Poking Gel in Sample Application Weak Bands Not Enough Sample Not Enough Stain Other Causes of Irregular and Distorted Bands Hemolyzed Sample Bent or Dirty Applicator Air Bubbles in Sample Application Too Much Sample Wick Flow Too Much Heat or Drying | View Page |
| Which statements below are associated with electroendosmosis? | View Page |
| Wick flow is caused by movement of the buffer ions into the medium when there has been a loss of moisture in the medium due to heat generation. | View Page |
| Electrophoresis and Molecular Diagnostics Because of ionized phosphate groups, both DNA and RNA will migrate in an electrical field with an appropriate buffer. They are negatively charged and will migrate to the anode. The speed of migration and separation achieved is based upon size with smaller molecules traveling faster. The shape of macromolecules, type of support medium, and electrophoresis method also vary the separation results. The isolated nucleic acid can be single-stranded or double-stranded and can fold into other structures. AGE, PAGE, and CE are the most common electrophoresis methods used in analysis of nucleic acids. Pulsed electric fields are needed to separate large fragments. The electrophoresis employed in blotting techniques enhance these discrimination techniques. | View Page |