Centrifugation Techniques and Chromatography
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Centrifugation Techniques and Chromatography

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Questions and Answers

What does isopycnic centrifugation primarily separate based on?

  • Weight of particles
  • Size of particles
  • Buoyant density of particles (correct)
  • Shape of particles

    • Which substance is commonly used to create a density gradient in isopycnic centrifugation?

  • Gelatin
  • Sucrose (correct)
  • Chitosan
  • Agarose

    • How does rate zonal centrifugation differ from isopycnic centrifugation?

  • It is unaffected by gravitational forces.
  • It distinguishes based on buoyant density.
  • It separates particles differing in size only. (correct)
  • It focuses on molecular weight instead of size or density.

    • In molecular exclusion chromatography, what defines the 'excluded volume'?

    <p>The volume of aqueous phase outside the beads</p> Signup and view all the answers

    What happens to very large size protein molecules during molecular exclusion chromatography?

    <p>They travel down the column rapidly.</p> Signup and view all the answers

    What is the purpose of washing and equilibrating the gel material in molecular exclusion chromatography?

    <p>To swell beads and achieve uniform pore size</p> Signup and view all the answers

    What is the primary measurement unit for buoyant density?

    <p>g/ml</p> Signup and view all the answers

    Why are small proteins retarded in a molecular exclusion chromatography column?

    <p>They penetrate the beads and travel in the pores.</p> Signup and view all the answers

    During which phase do submicroscopic protein aggregates begin to form?

    <p>Nucleation phase</p> Signup and view all the answers

    What initiates the protein precipitation process?

    <p>Addition of a precipitating agent</p> Signup and view all the answers

    What largely influences the concentration of salt needed for protein precipitation?

    <p>Number and distribution of charges and hydrophobic residues</p> Signup and view all the answers

    What happens to protein solubility as salt concentration increases?

    <p>It decreases</p> Signup and view all the answers

    In differential centrifugation, how are organelles primarily separated?

    <p>Based on their weight</p> Signup and view all the answers

    What is the result of the dehydration of proteins during the salting-out process?

    <p>Promotion of self-association and aggregation</p> Signup and view all the answers

    What is the primary purpose of centrifugation in biological studies?

    <p>To isolate pure samples of individual cell organelles</p> Signup and view all the answers

    Which of the following is NOT a substance mentioned that can be used to create a density gradient?

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

    What is the purpose of lysing the bacteria in the protein isolation process?

    <p>To remove soluble proteins from insoluble materials</p> Signup and view all the answers

    What is one consequence of continuing to centrifuge a sample beyond the necessary time?

    <p>All components will eventually form a pellet</p> Signup and view all the answers

    What happens to organelles during velocity centrifugation?

    <p>They separate based on their sedimentation rates</p> Signup and view all the answers

    At what critical size do protein particles stop growing through collision and flocculation?

    <p>0.1 µm to 10 µm</p> Signup and view all the answers

    What role does the density gradient play in density gradient centrifugation?

    <p>It maintains organelles in tight bands</p> Signup and view all the answers

    How does the activity of the protein of interest change with increasing concentrations in the supernatant?

    <p>It increases</p> Signup and view all the answers

    Which centrifugation technique separates organelles primarily based on their density?

    <p>Density gradient centrifugation</p> Signup and view all the answers

    Which is true regarding the order of separation in differential centrifugation?

    <p>Heaviest organelles are separated with low speeds</p> Signup and view all the answers

    What is the primary mechanism by which moving-boundary electrophoresis separates compounds?

    <p>Based on electric charge</p> Signup and view all the answers

    What measurement is used to detect the movement of compounds in moving-boundary electrophoresis?

    <p>Schlieren optics</p> Signup and view all the answers

    What is the purpose of a purification table in enzyme purification?

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

    How is the isoelectric pH of a protein typically determined using moving-boundary electrophoresis?

    <p>By determining electrophoretic mobility at several pH values</p> Signup and view all the answers

    What components are included in the moving-boundary electrophoresis apparatus?

    <p>A U-shaped cell and electrodes</p> Signup and view all the answers

    Which type of chromatography is based on specific binding properties?

    <p>Affinity chromatography</p> Signup and view all the answers

    What does the degree of purification in an enzyme purification process indicate?

    <p>The fold change in purity relative to crude extract</p> Signup and view all the answers

    In moving-boundary electrophoresis, what is the direction of compound migration based on?

    <p>The charge of the compounds</p> Signup and view all the answers

    What is the primary purpose of the low ionic strength buffer used in ion exchange chromatography?

    <p>To maintain protein stability</p> Signup and view all the answers

    In ion exchange chromatography, what happens to proteins that are charged oppositely to the ion-exchange media during the application step?

    <p>They are retained temporarily in the column</p> Signup and view all the answers

    Which method can be used to elute proteins from the column aside from increasing ionic strength?

    <p>pH change</p> Signup and view all the answers

    What is the main difference between adsorption chromatography and ion exchange chromatography?

    <p>Adsorption chromatography includes both positive and negative charged groups</p> Signup and view all the answers

    In adsorption chromatography, how does the binding strength to the stationary phase affect the movement of molecules?

    <p>Greater binding strength causes increased separation efficiency</p> Signup and view all the answers

    Which material is commonly used as a stationary phase in adsorption chromatography?

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

    What characteristic of silica makes it suitable for use as a stationary phase in chromatography?

    <p>It is acidic in nature</p> Signup and view all the answers

    How does the migration rate of proteins in electrophoresis relate to their physical characteristics?

    <p>It is governed by both the proteins' characteristics and the electrophoresis system</p> Signup and view all the answers

    Study Notes

    Centrifugation Techniques

    • Centrifugation isolates pure samples of cell organelles for biochemical study.
    • Differential centrifugation separates organelles based on weight; heaviest first at low speeds, lighter at high speeds.
    • Density gradient centrifugation separates organelles based on density using sucrose solutions layered in a test tube.
    • Velocity (rate-zonal) centrifugation uses a sucrose gradient to create bands of components based on sedimentation speed without separating them by buoyancy.
    • Equilibrium (isopycnic) centrifugation separates components by buoyant density, an equilibrium method where components remain stable once reached.

    Molecular Exclusion Chromatography

    • Uses hydrated polymer materials like dextran and agarose to prepare the column for separating proteins based on size.
    • Larger proteins are excluded from entering pores, moving faster through the column; smaller ones diffuse into the beads and travel slower.
    • Initial mixing involves adding a precipitating agent, leading to nucleation and aggregation of protein particles until a stable size is reached.
    • Salting out relies on salt concentration to precipitate proteins by reducing solubility and encouraging self-association.

    Protein Isolation Process

    • For isolating a novel protein: bacteria cultured, harvested by centrifugation, lysed using freeze/thaw, followed by centrifugation to collect soluble proteins.
    • Assays measure biological activity with a color change correlated to protein concentration.

    Ion Exchange Chromatography Steps

    • Proteins are transferred into a low ionic strength buffer and applied on a pre-equilibrated column with the same buffer.
    • Oppositely charged proteins are retained, while non-adhering proteins pass through.
    • Bursts in ionic strength gradually elute retained proteins by increasing net positive or negative charges.

    Adsorption Chromatography

    • Characterized by separating components based on their adsorption to the stationary phase within the column.
    • The efficiency of separation depends on solubility in the mobile phase and binding strength to the stationary phase (e.g., alumina or silica).
    • Incorporates both positive and negative charges, affecting protein migration and separation.

    Electrophoresis Techniques

    • Electrophoresis separates proteins in an electric field, migration rate influenced by the system and protein characteristics.
    • Moving-boundary electrophoresis uses a cell with buffer and electrodes to detect separation via refractive index changes at compound boundaries.
    • Isoelectric focusing determines the isoelectric pH at which proteins have no net charge, aiding in their identification.

    Affinity Chromatography

    • Separates proteins based on specific binding properties; highly selective for target proteins.

    Purification Tables

    • Purification tables track enzyme purification progress, listing total activity, total protein, specific activity, fold purification, and percentage recovery.
    • Each step indicates quantities to monitor yield and relative purity at each purification phase.

    Example Enzyme Purification

    • Calculating specific activity, percent yield, and degree of purification for an enzyme like acid phosphatase helps identify the most effective purification step based on fold increase.

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    Description

    This quiz covers essential techniques in centrifugation and molecular exclusion chromatography. Learn how differential, density gradient, velocity, and equilibrium centrifugation methods isolate cell organelles, as well as the principles behind size-based protein separation in chromatography. Test your knowledge on these critical laboratory methods.

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