Working with Proteins: Purification and Study
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Questions and Answers

What is one of the factors to consider when stabilizing isolated proteins?

  • Storing proteins at high temperatures
  • Blocking degradative enzymes such as nucleases & proteases (correct)
  • Reducing pH levels
  • Increasing adsorption to surface

    • Which technique is NOT used in protein purification according to the text?

  • Salting out
  • Electrophoresis
  • Chromatography
  • Ionization (correct)

    • What happens when salt is added during the 'salting in' process of protein solubility?

  • Protein solubility increases as salt is added (correct)
  • Additional ions intensify attractive forces between proteins
  • Proteins precipitate at lower salt concentrations
  • Solubility of protein decreases due to shielded charges

    • What is the most commonly used reagent for salting out proteins?

    <p>Ammonium sulfate</p> Signup and view all the answers

    How are proteins fractionated according to solubility in the text?

    <p>By molecular size</p> Signup and view all the answers

    Which enzyme-linked assay technique is mentioned in the text for assaying proteins?

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

    What happens to the solubility of a protein when its net charge is zero?

    <p>It is least soluble</p> Signup and view all the answers

    In chromatography, what is the purpose of the mobile phase?

    <p>To be a liquid for fractionation</p> Signup and view all the answers

    Which type of chromatography separates molecules based on size and shape?

    <p>Gel filtration chromatography</p> Signup and view all the answers

    What type of matrix is frequently used in ion-exchange chromatography?

    <p>Cellulose and agarose-based resin</p> Signup and view all the answers

    How are proteins eluted from an ion exchanger in chromatography?

    <p>By using a buffer with higher salt concentration or pH that reduces affinity to matrix</p> Signup and view all the answers

    What is monitored in the column effluent during ion-exchange chromatography?

    <p>Absorbance spectroscopy at 280 nm</p> Signup and view all the answers

    What is the purpose of affinity chromatography?

    <p>To estimate the mass of unknown proteins</p> Signup and view all the answers

    In affinity chromatography, how are proteins separated from other substances?

    <p>By washing the column with a high concentration of free ligand</p> Signup and view all the answers

    What is the role of pH in electrophoresis of proteins?

    <p>To separate proteins based on their charges</p> Signup and view all the answers

    How are separated amino acids made visible in paper electrophoresis?

    <p>By coating the paper with ninhydrin</p> Signup and view all the answers

    What is the purpose of SDS-PAGE in protein separation?

    <p>To separate proteins according to molecular mass</p> Signup and view all the answers

    Why is Coomassie blue used in electrophoresis of proteins?

    <p>To visualize separated proteins bands</p> Signup and view all the answers

    How does SDS bind to proteins?

    <p>By hydrophobic interaction</p> Signup and view all the answers

    What property of proteins does SDS-PAGE estimate?

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

    How are proteins separated in isoelectric focusing?

    <p>According to their isoelectric point</p> Signup and view all the answers

    What forms the pH gradient in isoelectric focusing?

    <p>Low molecular mass oligomers bearing amino and carboxylic acid groups</p> Signup and view all the answers

    In protein sequencing, how can the number of different subunits be determined?

    <p>By identifying the end group residues</p> Signup and view all the answers

    What does the equal amount of Glycine and Phenylalanine in insulin indicate?

    <p>Insulin contains two nonidentical polypeptide chains</p> Signup and view all the answers

    What is the key feature of using 1-fluoro-2,5-dinitrobenzene (FDNB) in protein sequencing for N-terminal identification?

    <p>Formation of a yellow color for easy visualization</p> Signup and view all the answers

    Which enzyme is typically used for C-terminal identification in protein sequencing?

    <p>Carboxypeptidase A</p> Signup and view all the answers

    What is the function of dansyl chloride in protein sequencing for N-terminal identification?

    <p>Intense yellow fluorescence for precise identification</p> Signup and view all the answers

    Which statement about carboxypeptidase A is correct?

    <p>Does not cleave C-terminal Arg or Lys residues</p> Signup and view all the answers

    How does aminopeptidase contribute to protein sequencing?

    <p>Liberates amino acids one at a time from the N-terminus</p> Signup and view all the answers

    What caution is provided regarding carboxypeptidase hydrolysis in protein sequencing?

    <p>Cleavage of the first residue slowly &amp; the second quickly suggests two different polypeptides</p> Signup and view all the answers

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