The movement of charged particles in an eletrical field. Useful in separating complex protein molecules into smaller fractions.
Why is Electrophoresis important in the lab?
It is used to detect, help diagnose, and monitor the diseases and conditions. Examples: Multiple meyloma (Protein), Isoenzymes, Hemoglobinopathies and thalassamia (Hb)
What is Iontophoresis
It is similar to electrophoresis, except it uses small ions rather than large proteins.
What are the three components of Electrophoresis?
Electric field
Charged particle (DNA, Protein)
Medium for movement to happen in (solid, liquid)
Electrophoresis Theory
Explain why and how ionized molecules can move through the medium.
Ionized molecule has an electrical charge
Moves toward either a cathod (-) or anode (+) depending on the charge of the molecule.
Positive ions move to cathode (-).
Negative ions move to anode (+).
What is an Ampholyte?
An ampholyte is a molecule that can be either negative or positive, depending on the pH of the solution it's on. Which way it moves depends on whether the solution is more acidic or more alkaline than the ampholyte's pI.
isoelectric point
The isoelectric point is the pH at which it has NO charge, and so it won't move in an electric field. Point where the ampholyte stops in electrophoresis.
In solution that's more acidic than it's pI, an ampholyte will ______ protons, take on a positive charge, and migrate toward the _________.
A.
In solutions that's more alkaline than it's pI, an amphylyte will ______ protons, take on a negative charge and migrate toward the ________.
A.
True or false: Proteins and Carbohydrates are ampholytes.
False: Proteins and nucleic acids
Rate of migration during electrophoresis depend on 5 factors... what are they.
Molecule's (protein/nucleic acid) charge
size and shape (Inversley proportional to rate)
strength of electric field (directly proportional to the rate)
medium properties (Inversley proportional to the rate)
Temperature
True or false: On gels, proteins are seperated in liquid matrix.
False: Solid matrix.
True or false: Greater staining intensity indicates higher protein concentration.
True
What type of medium supports zone electrophoresis (liquid or solid?), and why is it called zone?
Solid medium.Constituents separate into zones.
What are the five components of gel (zone)electrophoresis?
Electrical power
support medium
buffer
sampled
detection system
Electrophoresis procedure. Is this procedure in the right order?
1. Prepare reagents
2. Prepare gel
3. Add samples
4. Visualization of bands
5. Electrophoresis
No, 4 and 5 are switched.
True or false: Agarose gel has a high affinity for proteins.
False it has a low affinity. Neutral gel. It is made from agar.
This reagent of electrophoresis carries the electric current, establishes pH for the system, and resists the change of pH.
Buffer
Endosmostic flow / Endosmosis
It is the movement caused by the water in the electrophoresis system. Clouds of positive ions cluster around the immobolized hydroxl ions from water.
Electrokinetic potential
The potential between the fixed ions and the cloud of moveable ions. When electrical current is applied, the ions in the cloud move to the oppositely charged electrodes, taking water along. Macromolecules can be swept along with it.
Wick flow
Heat generated during electrophoresis causes evaporation of solvent, and buffer can rise up onto the support medium.
This affects migration and mobility of the gel.
True or false: Slab gel electrophoresis are popular with DNA/nucleic acid samples.
True
Disc Electrophoresis
Not used in hospital labs due to complexity. It involves the use of two gels of different concentrations stacked together. Disc means discontinous.
Isoelectric focusing
THis is a type of zone electrophoresis. It seperates the samples according to pH. Proteins migrates to pH region equal to its isoelectric point.
Densitometry
E.
Hospital labs use this type of electrophoresis since it's fast. ( 12,000 volts). Seperation takes place inside a small glass capillary.
e. disc. electrophoresis
C.
Immunofixation electrophoresis
The method detects by immunoprecipitation: when a soluble antigen (Ag) is brought in contact with the corresponding antibody, precipitation occurs, which may be visible with the naked eye or microscope. Immunofixation identifies (antibodies) in a mixture in function of their specific electrophoretic mobility. For the identification antigens are used that are specific for the targeted antibody.
A reduction technique is applied on a serum sample that has abnormal levels of serum/immunoglobulin. The abnormal peak will "disappear" after applying the anti-serum or anti-Immunoglobulin.
