Electrophoresis Techniques Quiz (CP508)
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Questions and Answers

What is the primary principle behind the ELISA technique?

  • Enzyme inhibition
  • Lipid membrane disruption
  • Antigen-antibody interaction (correct)
  • Protein denaturation

    • What role does the substrate play in an ELISA test?

  • It degrades antigens
  • It serves as an enzyme inhibitor
  • It undergoes a color change due to enzyme activity (correct)
  • It binds to antibodies

    • Which type of antibodies are best suited for fine detection and quantitation in ELISA?

  • Polyclonal antibodies
  • Monoclonal antibodies (correct)
  • Natural antibodies
  • Synthetic antibodies

    • Which enzyme labels are commonly used in ELISA?

    <p>Horseradish peroxidase and alkaline phosphatase</p> Signup and view all the answers

    In the context of ELISA, what is an antigen?

    <p>A substance that stimulates an immune response</p> Signup and view all the answers

    How are the antibodies typically linked to enzymes in an ELISA?

    <p>Via conjugation</p> Signup and view all the answers

    What distinguishes a direct ELISA from an indirect ELISA?

    <p>Whether the antigen or antibody is coated to the plate</p> Signup and view all the answers

    What is an enzyme conjugate in the context of ELISA?

    <p>An antibody linked to an enzyme</p> Signup and view all the answers

    What is a main advantage of using sandwich ELISA?

    <p>It has high specificity due to the use of two antibodies.</p> Signup and view all the answers

    In competitive ELISA, what does a higher concentration of antigen in the sample indicate?

    <p>Lower absorbance after the reaction.</p> Signup and view all the answers

    Which step occurs after the initial washing of the plate in the sandwich ELISA procedure?

    <p>Add enzyme-linked antibodies specific for the antigen.</p> Signup and view all the answers

    What is a crucial aspect of the procedure for competitive ELISA?

    <p>Antibody is initially incubated with the sample containing antigen.</p> Signup and view all the answers

    What might be a consequence of using fewer labeled antibodies in testing?

    <p>Cost savings associated with the testing procedure.</p> Signup and view all the answers

    What role does the substrate play in the sandwich ELISA procedure?

    <p>It is converted by the enzyme to form a colored product.</p> Signup and view all the answers

    What condition is necessary for the antigen-containing sample during sandwich ELISA incubation?

    <p>It must be incubated at 37°C.</p> Signup and view all the answers

    What happens to unbound enzyme-linked antibodies in the sandwich ELISA?

    <p>They are removed by washing.</p> Signup and view all the answers

    What is the main purpose of preparing several dilutions of standards starting from 5 mg/mL?

    <p>To create a calibration curve for concentration determination</p> Signup and view all the answers

    Which field is NOT mentioned as having applications for HPLC?

    <p>Astronomical applications</p> Signup and view all the answers

    What is one of the significant advantages of HPLC mentioned?

    <p>Needs a small sample with a high accuracy</p> Signup and view all the answers

    What is the primary basis for separation in Native-PAGE?

    <p>Charge, size, and shape</p> Signup and view all the answers

    Which component is NOT part of a UV-visible-NIR spectrophotometer?

    <p>Sample heater</p> Signup and view all the answers

    In which application would HPLC be used to determine the concentration of cocaine in blood?

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

    What role does SDS play in SDS-PAGE?

    <p>It provides a constant charge-mass ratio</p> Signup and view all the answers

    What type of sample analysis does a spectrophotometer perform?

    <p>None-destructive measurement of light intensity</p> Signup and view all the answers

    What happens to proteins when SDS is added during electrophoresis?

    <p>They gain negative charges proportional to their length</p> Signup and view all the answers

    What is a significant disadvantage of HPLC mentioned?

    <p>High solvent consumption</p> Signup and view all the answers

    What is a disadvantage of PAGE methods?

    <p>Electro osmotic effect</p> Signup and view all the answers

    What aspect of the sample analysis does the peak area or peak height relate to?

    <p>Concentration of the substance</p> Signup and view all the answers

    In terms of gel consistency, how does starch behave when prepared in a buffer?

    <p>It forms a clear colloidal suspension upon boiling</p> Signup and view all the answers

    What distinguishes thin layer electrophoresis from other electrophoresis methods?

    <p>It can be performed on thin layers of silica or alumina</p> Signup and view all the answers

    What is a notable advantage of thin layer electrophoresis?

    <p>It is less time consuming</p> Signup and view all the answers

    What is the effect of using petroleum jelly in the preparation of starch for electrophoresis?

    <p>Maintains the gel's shape and consistency</p> Signup and view all the answers

    What does the Beer-Lambert law relate to in a spectrophotometer?

    <p>The relationship between absorbance, path length, and concentration</p> Signup and view all the answers

    Which light source is typically used for the visible spectrum in spectrophotometers?

    <p>Tungsten lamp</p> Signup and view all the answers

    Which component in a spectrophotometer is responsible for dividing polychromatic light into its component wavelengths?

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

    What is the common material for cuvettes used in the ultraviolet region?

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

    What three factors influence the amount of light absorbed by a solution, according to the principles of a spectrophotometer?

    <p>Path length, concentration, and chemical nature</p> Signup and view all the answers

    Which type of monochromator commonly uses prisms?

    <p>Dispersion monochromators</p> Signup and view all the answers

    What is the role of the molar extinction coefficient (ε) in the Beer-Lambert law?

    <p>It is a constant for a specific molecule</p> Signup and view all the answers

    Which materials are suitable for constructing cuvettes for the infrared region?

    <p>Glass and quartz</p> Signup and view all the answers

    What is the primary advantage of the direct ELISA method?

    <p>It eliminates cross-reactivity of secondary antibodies.</p> Signup and view all the answers

    In which ELISA method is an antigen coated on the microtiter well and a secondary antibody used for detection?

    <p>Indirect ELISA</p> Signup and view all the answers

    What is the role of the substrate in the indirect ELISA process?

    <p>It is converted by the enzyme to produce a colored product.</p> Signup and view all the answers

    How does the sensitivity of indirect ELISA compare to direct ELISA?

    <p>Indirect ELISA is more sensitive because it uses multiple labeled antibodies.</p> Signup and view all the answers

    What step follows the addition of the sample containing the primary antibody in the indirect ELISA procedure?

    <p>Washing the plate to remove unbound secondary antibodies.</p> Signup and view all the answers

    What does the term 'cross-reactivity' refer to in the context of antibodies?

    <p>Antibodies reacting with non-target antigens.</p> Signup and view all the answers

    What is a critical consideration when using indirect ELISA to ensure accurate results?

    <p>Ensuring proper blocking of unbound sites.</p> Signup and view all the answers

    Which statement accurately reflects the procedure for competitive ELISA?

    <p>Antigen-antibody complexes are formed before binding takes place.</p> Signup and view all the answers

    Study Notes

    Practical Part of Spectral Analysis and Applied Spectroscopy (CP508)

    • Course offered at Cairo University, Faculty of Science, Chemistry Department
    • Intended for Biochemistry Diploma Students
    • First Semester, 2024-2025

    Electrophoresis Techniques

    • Electrophoresis is the migration of charged particles (DNA, RNA, protein) through a solution under an external electric field.

    • The rate of ion migration depends on:

      • Net charge of the molecule
      • Size and shape of the particle
      • Strength of the electric field
      • Properties of the supporting medium
      • Temperature of operation

    • Particle mobility is affected by particle size and shape, and the medium.

    Types of Electrophoresis

    • Zone Electrophoresis
      • Paper electrophoresis
      • Gel electrophoresis
      • Thin-layer electrophoresis
      • Cellulose acetate electrophoresis
    • Moving Boundary Electrophoresis
      • Capillary electrophoresis
      • Isotachophoresis
      • Isoelectric Focusing

    Paper Electrophoresis

    • Filter papers (Whatman No. 1 and 3) are used, in strips of 3mm or 5cm.
    • Separation takes 12-14 hours.
      • Advantages: economical, easy to use.
      • Disadvantages: certain compounds (proteins, hydrophilic molecules) cannot be resolved due to adsorptive and ionic properties of paper.

    Gel Electrophoresis

    • Separation is based on molecular sieving, using the molecular size of the substances.
    • Gel material (primarily water) acts as a molecular sieve.
    • Types include: Agar, Agarose, Polyacrylamide, Starch gels.

    Agar and Agarose Gel

    • Agar: Mixture of polysaccharides from seaweed.
    • Agarose: Highly purified polysaccharide from agar.
    • Advantages: Easy to prepare, High resolution.
    • Disadvantages: Electro osmosis is high, Resolution is less compared to polyacrylamide gels.

    Polyacrylamide Gel Electrophoresis (PAGE)

    • Prepared by polymerizing acrylamide monomers using methylene-bis-acrylamide for cross-linking.
    • Structures: CH2=CH-CO-NH2
    • Polyacrylamide gels: Tougher than agarose gels.

    HPLC (High-Performance Liquid Chromatography)

    • Separates mixtures of components based on different affinities for a mobile phase and a stationary adsorbent.
      • Components: Mixture, Separated Components, Analyze, Identify, Purify, Quantify
    • Explanation: A compound is placed on the stationary phase, and the mobile phase passes through, carrying components a certain distance, based on their attraction to both the phases.
    • Types:
      • Normal phase chromatography (uses a polar stationary phase and a non-polar mobile phase)
      • Reversed phase chromatography (uses a nonpolar stationary phase and a polar mobile phase)

    Spectrophotometer

    • Measures intensity of electromagnetic energy at different wavelengths.
    • Components: Light source, monochromator, detector.

    Enzyme-linked Immunosorbent Assay (ELISA)

    • A biochemical technique using antigen-antibody interaction for detecting and quantifying peptides, proteins, antibodies, enzymes, and hormones.
    • Components:
      • Antigen
      • Antibody
      • Enzyme conjugate
      • Substrate
    • Types:
      • Direct ELISA
      • Indirect ELISA
      • Sandwich ELISA
      • Competitive ELISA

    Flow Cytometry

    • Measures the physical and chemical properties of cells as they pass through a laser beam in a single file.
    • Components: Fluidics, Optics, Electronics
    • Applications: Cell health status, detection of apoptotic cells, identifying cell subsets.

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    Related Documents

    Practical Part of Spectral Analysis and Applied Spectroscopy (CP508) 2024-2025 PDF

    Description

    Test your knowledge on electrophoresis techniques as covered in the Practical Part of Spectral Analysis and Applied Spectroscopy course at Cairo University. This quiz will evaluate your understanding of various methods, principles, and factors affecting electrophoresis for biochemistry students.

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