Isoelectric Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Isoelectric and links to relevant pages within the course.
Learn more about laboratory continuing education for medical technologists to earn CE credit for AMT, ASCP, NCA, and state license renewal and recertification. Or get information about laboratory safety and compliance courses that deliver cost-effective OSHA safety training and continuing education to your laboratory's employees.
|Which of the following methods would be employed to collect sweat for chloride analysis:||View Page|
|Which of the following methods would be used to confirm the presence of Bence-Jones
protein in the urine:||View Page|
|Heat and Acid Test for Urinary Protein||View Page|
|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
|Isoelectric Point (pI)|
Isoelectric point (pI) is the pH where the net charge of a molecule is zero. At its pI, a molecule will not move in an electrical field because it does not have a charge. An amphoteric molecule such as a protein in a pH below its pI will have a net positive charge. Conversely, if the pH is above the pI, a protein will have a net negative charge.
|Which statement is correct for a protein with an isolectric point (pI) of 7.0?||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.
|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.
|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|
|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|
When hemoglobin S (HbS) has been detected using a primary screening method, it is then crucial to determine if the HbS is homozygous, heterozygous, or is another variant. Additional procedures that may be needed include isoelectric focusing (IEF) or high performance liquid chromatography (HPLC).Observation of RBC morphology and use of sickle cell screening may provide initial information for this diagnosis, but may be negative in some sickle cell disorders.Polymerized chain reaction (PCR) methods are being used in prenatal diagnosis of sickle cell disorders.