Electrophoresis Principles
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Questions and Answers

What is a primary advantage of moving boundary electrophoresis?

  • Requires denaturating agents
  • Biologically active fragments can be recovered (correct)
  • No need for external power supply
  • High resolution

    • What is a significant disadvantage of moving boundary electrophoresis?

  • Low resolution due to mixing of the sample (correct)
  • Does not allow for sample recovery
  • Inability to measure electrophoretic mobility
  • Requires a supporting media

    • Which equipment is essential in moving boundary electrophoresis?

  • Gel matrix
  • U shape tube (correct)
  • Chromatographic column
  • Spectrophotometer

    • Which type of electrophoresis does not require a supporting medium?

    <p>Moving boundary electrophoresis</p> Signup and view all the answers

    In moving boundary electrophoresis, which device measures the change in refractive index?

    <p>Refractometer</p> Signup and view all the answers

    What happens to charged molecules during moving boundary electrophoresis?

    <p>They move to their respective counter charge electrodes</p> Signup and view all the answers

    In Zone Electrophoresis, what happens to amphoteric substances initially at a pH below their isoelectric point?

    <p>They become positively charged and migrate towards the cathode.</p> Signup and view all the answers

    Which of the following statements about the pH gradient in Zone Electrophoresis is correct?

    <p>The pH continuously increases from anode to cathode.</p> Signup and view all the answers

    What type of supporting media is used in Gel Electrophoresis for the separation of high molecular weight substances?

    <p>Semi-solid gel</p> Signup and view all the answers

    Which of the following materials is NOT used in Gel Electrophoresis?

    <p>Silica gel</p> Signup and view all the answers

    In Gel Electrophoresis, what key property of the gel helps in the separation of high molecular weight substances?

    <p>Molecular sieving</p> Signup and view all the answers

    What role does SDS play in protein electrophoresis?

    <p>It confers a uniform negative charge to proteins.</p> Signup and view all the answers

    Why do smaller proteins move farther down the gel in SDS-PAGE?

    <p>They more easily fit through the pores in the gel.</p> Signup and view all the answers

    What happens when an electric current is applied across the gel in SDS-PAGE?

    <p>Negatively charged proteins migrate towards the positive pole.</p> Signup and view all the answers

    What is the purpose of the buffer in SDS-PAGE?

    <p>To provide uniform pH and ions for conducting electric potential.</p> Signup and view all the answers

    What is the effect of protein size on migration distance in SDS-PAGE?

    <p>Larger proteins stay closer to the point of origin.</p> Signup and view all the answers

    What does 'pheresis' refer to in the context of electrophoresis?

    <p>Migration</p> Signup and view all the answers

    Which of the following is NOT a factor that influences the rate of migration of molecules in electrophoresis?

    <p>The refractive index of the medium</p> Signup and view all the answers

    During electrophoresis, negatively charged molecules move towards which electrode?

    <p>Anode</p> Signup and view all the answers

    Which type of electrophoresis involves the solute moving through the supporting media?

    <p>Moving boundary electrophoresis</p> Signup and view all the answers

    What is the isoelectric point of a protein?

    <p>The pH at which the net charge of the protein is zero</p> Signup and view all the answers

    Which of the following is an advantage of moving boundary electrophoresis?

    <p>Addition of reference material</p> Signup and view all the answers

    Zone electrophoresis is known for its high resolution. What is the disadvantage of moving boundary electrophoresis in this regard?

    <p>Lower resolution</p> Signup and view all the answers

    Which of the following particles move towards the cathode in an electric field?

    <p>Cations</p> Signup and view all the answers

    Which component of the gel electrophoresis system is used to set up an electrical field?

    <p>Power Supply</p> Signup and view all the answers

    What is the primary factor used to separate molecules in gel electrophoresis?

    <p>Size and charge</p> Signup and view all the answers

    What is the function of the gel in gel electrophoresis?

    <p>To retard the passage of molecules according to their size and shape</p> Signup and view all the answers

    Which type of gel is commonly used for the separation of proteins?

    <p>Cellulose acetate</p> Signup and view all the answers

    Which gel type is typically used for the separation of nucleic acids?

    <p>Agarose</p> Signup and view all the answers

    What is the main purpose of adding loading dye to a sample in gel electrophoresis?

    <p>To make the sample visible for proper loading</p> Signup and view all the answers

    What keeps the gel at a constant temperature during electrophoresis?

    <p>Running Buffer</p> Signup and view all the answers

    What unique feature does vertical gel have in the separation process?

    <p>No technical issues</p> Signup and view all the answers

    What is the primary function of the running buffer in gel electrophoresis?

    <p>To carry the electrical current through the gel</p> Signup and view all the answers

    Where are the wells located in a gel electrophoresis setup?

    <p>Along the side of the gel closest to the negative electrode</p> Signup and view all the answers

    What is the purpose of the loading dye in gel electrophoresis?

    <p>To make the solution denser and color the sample</p> Signup and view all the answers

    Which characteristic is true for vertical gel boxes?

    <p>Used for polyacrylamide gel electrophoresis</p> Signup and view all the answers

    How does the running buffer help maintain the gel's integrity during electrophoresis?

    <p>By keeping the gel cool</p> Signup and view all the answers

    What ensures that the samples move through the middle of the gel during electrophoresis?

    <p>Application of current to the gel</p> Signup and view all the answers

    What is the primary use of a horizontal (flatbed) gel box?

    <p>Agarose gel electrophoresis</p> Signup and view all the answers

    In which step of the procedure for DNA separation should you ensure the gel is completely submerged in the buffer?

    <p>When placing the gel in the gel box</p> Signup and view all the answers

    What should be done immediately after the electrophoresis is complete?

    <p>Switch off the power supply</p> Signup and view all the answers

    Why is the horizontal gel box considered safer in terms of electricity accidents?

    <p>It has a limited gel thickness.</p> Signup and view all the answers

    Where should the wells be oriented in the gel box for proper electrophoresis?

    <p>Closest to the negative electrode</p> Signup and view all the answers

    What is the purpose of adding loading dye to the sample?

    <p>To monitor the progress of electrophoresis</p> Signup and view all the answers

    Which electrode should be connected to the positive end of the gel box?

    <p>The anode</p> Signup and view all the answers

    Which protein is typically found in the largest quantity in serum protein electrophoresis?

    <p>Albumin</p> Signup and view all the answers

    What is the first step in loading a sample onto the gel?

    <p>Add loading dye to the sample</p> Signup and view all the answers

    Which component of serum protein electrophoresis is responsible for transporting oxygen?

    <p>Albumin</p> Signup and view all the answers

    What is one of the purposes of using a standard in DNA gel electrophoresis?

    <p>To estimate the length of DNA strands in the sample</p> Signup and view all the answers

    Which of the following is a specific application of DNA gel electrophoresis in neoplastic disorders?

    <p>Analysis of monoclonality</p> Signup and view all the answers

    How are DNA standards treated in gel electrophoresis as compared to the samples?

    <p>DNA standards are treated the same way as samples</p> Signup and view all the answers

    Which of the following is not a specific application of DNA electrophoresis mentioned in the content?

    <p>Identification of RNA sequences</p> Signup and view all the answers

    What laboratory examination is commonly used to identify patients with multiple myeloma?

    <p>Serum protein electrophoresis</p> Signup and view all the answers

    Which of the following factors must be considered when estimating the length of a DNA band in a gel?

    <p>The position of other bands in the same gel</p> Signup and view all the answers

    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.

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