Questions and Answers
What is a primary advantage of moving boundary electrophoresis?
What is a significant disadvantage of moving boundary electrophoresis?
Which equipment is essential in moving boundary electrophoresis?
Which type of electrophoresis does not require a supporting medium?
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In moving boundary electrophoresis, which device measures the change in refractive index?
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What happens to charged molecules during moving boundary electrophoresis?
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In Zone Electrophoresis, what happens to amphoteric substances initially at a pH below their isoelectric point?
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Which of the following statements about the pH gradient in Zone Electrophoresis is correct?
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What type of supporting media is used in Gel Electrophoresis for the separation of high molecular weight substances?
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Which of the following materials is NOT used in Gel Electrophoresis?
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In Gel Electrophoresis, what key property of the gel helps in the separation of high molecular weight substances?
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What role does SDS play in protein electrophoresis?
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Why do smaller proteins move farther down the gel in SDS-PAGE?
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What happens when an electric current is applied across the gel in SDS-PAGE?
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What is the purpose of the buffer in SDS-PAGE?
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What is the effect of protein size on migration distance in SDS-PAGE?
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What does 'pheresis' refer to in the context of electrophoresis?
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Which of the following is NOT a factor that influences the rate of migration of molecules in electrophoresis?
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During electrophoresis, negatively charged molecules move towards which electrode?
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Which type of electrophoresis involves the solute moving through the supporting media?
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What is the isoelectric point of a protein?
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Which of the following is an advantage of moving boundary electrophoresis?
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Zone electrophoresis is known for its high resolution. What is the disadvantage of moving boundary electrophoresis in this regard?
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Which of the following particles move towards the cathode in an electric field?
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Which component of the gel electrophoresis system is used to set up an electrical field?
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What is the primary factor used to separate molecules in gel electrophoresis?
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What is the function of the gel in gel electrophoresis?
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Which type of gel is commonly used for the separation of proteins?
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Which gel type is typically used for the separation of nucleic acids?
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What is the main purpose of adding loading dye to a sample in gel electrophoresis?
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What keeps the gel at a constant temperature during electrophoresis?
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What unique feature does vertical gel have in the separation process?
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What is the primary function of the running buffer in gel electrophoresis?
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Where are the wells located in a gel electrophoresis setup?
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What is the purpose of the loading dye in gel electrophoresis?
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Which characteristic is true for vertical gel boxes?
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How does the running buffer help maintain the gel's integrity during electrophoresis?
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What ensures that the samples move through the middle of the gel during electrophoresis?
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What is the primary use of a horizontal (flatbed) gel box?
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In which step of the procedure for DNA separation should you ensure the gel is completely submerged in the buffer?
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What should be done immediately after the electrophoresis is complete?
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Why is the horizontal gel box considered safer in terms of electricity accidents?
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Where should the wells be oriented in the gel box for proper electrophoresis?
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What is the purpose of adding loading dye to the sample?
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Which electrode should be connected to the positive end of the gel box?
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Which protein is typically found in the largest quantity in serum protein electrophoresis?
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What is the first step in loading a sample onto the gel?
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Which component of serum protein electrophoresis is responsible for transporting oxygen?
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What is one of the purposes of using a standard in DNA gel electrophoresis?
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Which of the following is a specific application of DNA gel electrophoresis in neoplastic disorders?
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How are DNA standards treated in gel electrophoresis as compared to the samples?
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Which of the following is not a specific application of DNA electrophoresis mentioned in the content?
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What laboratory examination is commonly used to identify patients with multiple myeloma?
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Which of the following factors must be considered when estimating the length of a DNA band in a gel?
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Study Notes
Electrophoresis
- Electrophoresis is the movement of charged solutes or particles in a liquid medium under the effect of an electric field.
- Positively charged molecules (cations) move towards the negative pole (cathode), while negatively charged molecules (anions) move towards the positive pole (anode).
- The rate of migration through the electrical field depends on:
- The strength of the field
- The net charge, size, and shape of the molecules
- The ionic strength, viscosity, and temperature of the medium
Applications of Electrophoresis
- Separation of biological molecules such as amino acids, nucleic acids, proteins, peptides, and organic acids/bases
- Analysis of charged species or mixtures
- Determination of the isoelectric point of proteins
Types of Electrophoresis
- Moving boundary electrophoresis
- Zone electrophoresis
- Iso-electric focusing (IEF)
Moving Boundary Electrophoresis
- Involves the movement of solutes through a supporting medium
- The molecules move to their respective poles under the electric field
- A refractometer measures the concentration of molecules during electrophoresis
- Advantages: reference material can be added to help identify active fragments
- Disadvantages: low resolution of the sample
Zone Electrophoresis
- Separation of amphoteric substances in an electric field depending on both voltage and pH gradients
- pH increases gradually from anode to cathode
- Substances that are initially at pH below their isoelectric point will be positively charged and migrate to the cathode, while those above their isoelectric point will be negatively charged and migrate to the anode
Gel Electrophoresis
- Uses gel as a supporting medium for separation of high molecular weight substances such as proteins and nucleic acids by molecular sieving
- Materials used: agar, agarose, polyacrylamide, and starch gel
- Semi-solid, water-insoluble gel
- Proteins are dissolved in SDS and electrophorized, resulting in uniform negative charge
- The gel acts as a sieve to retard the passage of molecules according to their size and shape
Features of Gel Electrophoresis
- Gel type
- Gel concentration
- Buffer type/pH
Components of Gel Electrophoresis System
- Power supply
- Gel box
- Running buffer
- Wells
- Loading dye
- Shapes of gel (vertical, horizontal)
Running Buffer
- Solution used to carry the electrical current through the gel
- Helps keep the gel cool
Wells
- Small indentations created in the gel when it is made
- Uniformly spaced along the side of the gel closest to the negative electrode
Loading Dye
- Colored buffer mixed with the material prior to loading onto the gel
- Makes the solution denser than the surrounding running buffer, allowing the sample to sink into the well
Shapes of Gel Boxes
- Vertical gel box: different gel thickness, more than one gel per apparatus, not easily adapted for different techniques
- Horizontal (flatbed) gel box: gel thickness limited, only one gel per apparatus, easily adapts different techniques, technician friendly
Procedure for Separation of DNA
- Prepare the gel box by adding running buffer
- Place the gel in the gel box and add loading dye to each sample
- Load the sample into the corresponding well
- Connect the electrodes to the power supply
- Run the electrophoresis
- Visualize the gel using a UV transilluminator
Specific Applications of Electrophoresis
- Neoplastic disorders: detection of tumor-related mutations, microsatellite instability, and analysis of monoclonality
- Diagnosis of hereditary diseases and prenatal testing
- Diagnosis of infectious diseases
- Identity testing, e.g. serum protein electrophoresis
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Description
Understand the basics of electrophoresis, including the movement of charged particles in a liquid medium and factors affecting migration rate.