Biochemistry Chapter on Rate-Zonal Centrifugation
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Questions and Answers

What is the primary technique used to determine the amino acid sequence of a protein before 1985?

  • Edman degradation (correct)
  • Nuclear magnetic resonance
  • X-ray crystallography
  • Mass spectrometry
  • What does Edman degradation rely on for amino acid identification?

  • Physical separation of proteins
  • Gene cloning techniques
  • Ionization of proteins
  • High-pressure liquid chromatography (correct)
  • What is the advantage of using recombinant DNA techniques over Edman degradation for sequencing proteins?

  • They directly analyze protein structure.
  • They are generally faster than chemical sequencing. (correct)
  • They can identify more amino acids at once.
  • They are more precise than chemical methods.
  • Which of the following steps is NOT involved in the Edman degradation process?

    <p>Ionizing the molecular sample (A)</p> Signup and view all the answers

    What is required to initiate the Edman degradation process?

    <p>Phenylisothiocyanate (PITC) (C)</p> Signup and view all the answers

    In the context of mass spectrometry, what role does the organic acid play?

    <p>It helps in the drying process of the sample. (D)</p> Signup and view all the answers

    What has contributed to the rapid expansion of genome sequence databases?

    <p>Development of DNA sequencing methods (B)</p> Signup and view all the answers

    How does the Edman degradation affect the polypeptide after each cycle?

    <p>It removes one amino acid from the N-terminus. (C)</p> Signup and view all the answers

    What is the primary purpose of isoelectric focusing (IEF)?

    <p>To create a pH gradient for protein separation (B)</p> Signup and view all the answers

    How do proteins migrate during isoelectric focusing?

    <p>According to their pI, or isoelectric point (B)</p> Signup and view all the answers

    In two-dimensional gel electrophoresis, what is the second dimension based on?

    <p>The molecular weight of the proteins (D)</p> Signup and view all the answers

    What advantage does two-dimensional gel electrophoresis have in studying cellular proteins?

    <p>It allows for the resolution of many proteins simultaneously (A)</p> Signup and view all the answers

    What does liquid chromatography separate molecules based on?

    <p>Interactions with a solid surface (D)</p> Signup and view all the answers

    Which of the following statements is true regarding the proteins during the SDS PAGE step?

    <p>They separate according to their mass. (C)</p> Signup and view all the answers

    What is the role of the isoelectric point (pI) in protein separation techniques?

    <p>It is the pH at which a protein has no net charge. (C)</p> Signup and view all the answers

    Why is isoelectric focusing effective for resolving proteins that differ by only one charge unit?

    <p>It establishes a precise pH gradient. (B)</p> Signup and view all the answers

    What is the primary function of rate-zonal centrifugation?

    <p>To separate molecules of interest into distinct bands (A)</p> Signup and view all the answers

    What can influence the sedimentation rate in rate-zonal centrifugation?

    <p>Both mass and shape of particles (A)</p> Signup and view all the answers

    In rate-zonal centrifugation, what happens if the centrifuge is operated for too long?

    <p>All molecules will form a pellet at the bottom (C)</p> Signup and view all the answers

    Which technique is mostly used for separating DNA or organelles?

    <p>Equilibrium density-gradient centrifugation (D)</p> Signup and view all the answers

    What is the main principle behind electrophoresis?

    <p>Molecules separate according to their charge:mass ratio (C)</p> Signup and view all the answers

    How does the charge of a molecule affect its speed during electrophoresis?

    <p>Greater net charge results in faster movement towards an electrode (D)</p> Signup and view all the answers

    What is one common use of electrophoresis?

    <p>To resolve small molecules like nucleotides (B)</p> Signup and view all the answers

    What factor is not relevant in determining how molecules migrate in electrophoresis?

    <p>Color of the molecule (C)</p> Signup and view all the answers

    What is the major site of DNA synthesis as identified by autoradiographic studies using [3H]thymidine?

    <p>Nucleus (D)</p> Signup and view all the answers

    After prolonged incubation with [3H]thymidine, where does most of the radioactivity remain?

    <p>In the nucleus (A)</p> Signup and view all the answers

    What does the localization of autoradiographic grains over the cytoplasm indicate about RNA?

    <p>RNA is transported from the nucleus (A)</p> Signup and view all the answers

    How long are the tracks created by the β particles emitted by tritium in autoradiographic studies?

    <p>0.47 μm (C)</p> Signup and view all the answers

    What is the approximate accuracy of locating 3H-labeled structures within cells?

    <p>0.5–1.0 mm (A)</p> Signup and view all the answers

    What factor significantly affects the localization ability of autoradiography?

    <p>The energy of particles emitted during radioactive disintegrations (C)</p> Signup and view all the answers

    Which of the following statements about the emission of β particles by phosphorus-32 is true?

    <p>They generate very long streaks on photographic emulsion. (B)</p> Signup and view all the answers

    In autoradiographic studies using [3H]uridine, where are the grains predominantly located after a long incubation period?

    <p>In both the nucleus and cytoplasm (C)</p> Signup and view all the answers

    What is the primary use of x-ray diffraction in protein analysis?

    <p>To establish the detailed three-dimensional structure of proteins (D)</p> Signup and view all the answers

    What challenge is associated with crystallizing large multisubunit proteins?

    <p>They often require precise conditions that are difficult to replicate (D)</p> Signup and view all the answers

    How are the x-rays used in diffraction experiments to analyze protein structures?

    <p>They form a scattering pattern when passing through a crystal (D)</p> Signup and view all the answers

    What is a key advantage of cryoelectron microscopy compared to x-ray crystallography?

    <p>It does not require crystallization of proteins (D)</p> Signup and view all the answers

    Which wavelengths are used in x-ray diffraction studies?

    <p>0.1–0.2 nanometers (B)</p> Signup and view all the answers

    What role do heavy metal modifications play in x-ray crystallography?

    <p>They aid in interpreting the diffraction pattern (D)</p> Signup and view all the answers

    What is the outcome of scattering x-rays in x-ray diffraction experiments?

    <p>A diffraction pattern containing discrete spots (B)</p> Signup and view all the answers

    What is a critical factor to consider when using electron microscopy for protein analysis?

    <p>Radiation should be minimized to prevent structural damage (C)</p> Signup and view all the answers

    Study Notes

    Rate-Zonal Centrifugation

    • Proteins are separated into bands or disks in a tube based on mass using rate-zonal centrifugation.
    • Samples must be centrifuged for optimal time; too short leads to insufficient separation, while too long results in pellet formation.
    • Sedimentation rates are influenced by both mass and shape, complicating precise molecular weight determination.
    • Rate-zonal centrifugation remains practical for separating varied polymers and particles.

    Equilibrium Density-Gradient Centrifugation

    • Primarily used for separating DNA or organelles.
    • Employs a density gradient to allow different components to settle at their respective densities.

    Electrophoresis Techniques

    • Electrophoresis separates molecules in mixtures using an electric field, influenced by their charge:mass ratio.
    • Molecules with the same mass and shape migrate at different speeds based on net charge.
    • Isoelectric focusing (IEF) resolves proteins by migrating them through a pH gradient until they reach their isoelectric point (pI).

    Two-Dimensional Gel Electrophoresis

    • Combines IEF with SDS electrophoresis for dual separation based on charge and mass.
    • Allows up to 1000 proteins to be resolved simultaneously, aiding in gene expression studies.

    Liquid Chromatography

    • Separates proteins, nucleic acids, and other molecules by their interaction with a solid surface in a liquid medium.
    • Critical for autoradiographic studies, identifying synthesis sites of molecules within cells, like DNA in the nucleus and RNA movement to the cytoplasm.

    Autoradiography

    • Involves using a radiation-sensitive photographic emulsion over labeled cells to visualize radioactive incorporation sites.
    • Emission energy of isotopes impacts localization accuracy; e.g., tritium offers better resolution than phosphorus-32.

    Edman Degradation

    • Classic method for amino acid sequencing; targets the N-terminus of polypeptides for sequential identification.
    • Involves labeling, cleavage, and identification, allowing for gradual reduction of the polypeptide chain.

    Recombinant DNA Techniques

    • Offer faster methods for deducing protein sequences from mRNA or gene sequences than Edman degradation.
    • Genome sequencing advancements enhance protein research and understanding of large proteins.

    Mass Spectrometry

    • Measures mass of proteins and peptides through ionization, acceleration, and detection of molecular ions.
    • Essential for establishing three-dimensional structures of proteins via x-ray diffraction patterns.

    Cryoelectron Microscopy

    • Utilized for proteins that do not crystallize easily; captures low-resolution images of proteins in a frozen state.
    • The technique involves rapid freezing and electron microscopy, with computer analysis for three-dimensional structure reconstruction.

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    Biological Macromolecules PDF

    Description

    Explore the principles behind rate-zonal centrifugation, a key technique in biochemistry for separating proteins based on their masses. Understanding the balance of centrifugation time is crucial for achieving effective separation. Test your knowledge about this density-gradient method and its applications in protein analysis.

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