Lecture Three - Purification Techniques

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Lecture Three - Purification Techniques
2013-09-21 17:05:44
Biochemistry Lecture Three Purification Techniques

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  1. What makes Purification difficult?
    • Many constituents of the cell have similar physiological characters.
    • Materials may be unstable outside of the cell.
    • Materials may be present in extremely small quantities.
  2. What is a key driving force behind Biochemical advancement?
    Development of new purification techniques
  3. What are the two biggest purification tools in Biochemistry?
    Chromatography and Electrophoresis.
  4. What are the Five main Physical Properties that are used to purify materials?
    • Solubility
    • Ionic Charge
    • Polarity
    • Molecular Size
    • Binding Specificity
  5. Give an example of Purification by Solubility.
    Salting in -> Salting out
  6. Give example(s) of Purification by Ionic Charge.
    • Ion exchange Chromatography -> Electrophoresis
    • Isoelectric focusing (Electrophoresis)
  7. Give example(s) of Purification by Polarity.
    • Adsorbtion Chromatography -> Paper Chromatography
    • Reverse-Phase Chromatography -> Hydrophobic Interaction Chromatography
  8. Give example(s) of Purification by Molecular Size.
    • Dialysis & Ultrafiltration -> Gel electrophoresis.
    • Size exclusion Chromatography -> Ultrafiltration
  9. Give an example of Purification by binding specificity.
    Affinity chromatography.
  10. Which specific Purification Technique is the most powerful? What is it's downfall?
    Affinity Chromatography is the most powerful technique however it cannot be applied to all systems.
  11. When/how is Solubility-based purification used?
    As a primary purification technique seeing as it isn't quite perfect.
  12. What kinds of things affect Protein solubility?
    • Ionic strength
    • Organic Solvents
    • pH
    • Etc...
  13. Briefly describe how you might conduct a Solubility-based purification.
    • Adjust a physiochemical property (such as salinity) to just below the point at which the protein of interest precipitates.
    • (Contaminating proteins of lesser solubility will have precipitated).
    • Centrifuge and/or decant off supernatant
  14. Give to physiological conditions (discussed in class) you might alter to purify by solubility.
    • Ionic Concentration (Salinity)
    • Temperature (for purifying thermophile proteins)
  15. How might you purify thermophile proteins from a non-thermophillic host?
    • Raise the temperature to ~60c.
    • All non thermophillic proteins will denature and precipitate.
    • Remove supernatant containing thermophillic proteins.
  16. Brlow is a graph of Solubility (of a protein) vs Ionic Strength. What do the letters I, O and M stand for?
    • I - Salting In
    • O- Salting Out
    • M - Maxima
  17. Describe the graph shown below.
    The Solubility of Proteins increases along with Ionic strength until a Maxima is reached. Then Solubility decreases with Ionic Strength
  18. Briefly explain the graph below.
    At Low salt concentrations (region I) there is enough available water in the solution to negate like-charges of the proteins allowing closer packing and better packing.

    As salt concentrations increase, a point is reached (M) where the water has become more scarce. Therefore, the salt begins "stealing" water from the dissolved proteins, reducing their solubility (region O).
  19. Why does salt effectively "steal" water from proteins at high concentrations?
    Each salt molecule (e.g MgCl) has at least one full charge associated with it's relatively small size. Proteins may or may not have full charges and have a much larger size. Therefore the Ionic Solute (salt) has a much stonger "pull" on the limited water causing it to "steal" it.
  20. What is it called when like charges are negated by some other (usually smaller) molecule (E.g Salting in)?
  21. What is Affinity Chromatography?
    Using a specific matrix to bind a specific protein.
  22. How does Affinity Chromatography work?
    • A Ligand is Covalently Bonded to an inert, porous matrix.
    • The protein will tightly bind non-covalently to the Ligand.
  23. Why is Affinity Chromatography so useful?
    It often gives yields of up to 99% pure protein.
  24. How is protein bound to an Affinity Column recovered?
    • Altering Elution conditions such as Ionic Strength, pH etc.
    • Another way is to flush the column with ligand which will bind to the protein in place of the matrix-based substrate (so long as the buffer substrate is higher in conc.).
  25. Nickel Affinity Chromatography is an Example of which type of Chromatography?
    Affinity Chromatography.
  26. How does Nickel Affinity Chromatography work?
    • The Protein is synthesized with a Histidine Tag
    • Then the protein (and other stuff) is put through a nickel column
    • The Histidine Tag Binds tightly to Nickel separating it from the other "stuff".
  27. Why is Nickel so effective in Chromatography?
    It is rarely found in Biology.
  28. How do you elute the Nickel-column after separation?
    Use a Histidine-like substance.
  29. What does HPLC stand for?
    High Performance Liquid Chromatography
  30. What is HPLC?
    Using a force to draw the Mobile Phase through the Chromatography Column.
  31. In what ways is HPLC an improvement of Chromatography?
    • It allows for a greater number of smaller beads (Improved Separation).
    • It has a greater sensitivity (Narrower Peaks)
    • It can be Automated
  32. How is a force applied to the Mobile Phase of a Chromatography Column?
    Using pumps that generate up to 5000psi in force.
  33. To which Chromatography Methods does HPLC apply?
    To all of them.
  34. What is the definition of Electrophoresis?
    The Migration of Ions through an Electric Field.
  35. Electrophoresis is most effective at,
    A) Seperation
    B) Purification
    C) Ionization
    C) None of the Above
    • A) Seperation
    • Electrophoresis can only be used for small quantities of material so is not particularly useful in Purification.
  36. Chromatography is most effective at,
    A) Seperation
    B) Purification
    C) Ionization
    C) None of the Above
    • B) Purification
    • Chromatography is effective for separating large amounts of material but is not as good (for separation) as Electrophoresis.
  37. What affects Migration of Ions in Electrophoresis?
    • Charge (q)
    • Frictional Coefficient (f)
  38. The coefficient f is dependant on a number of factors. What are they?
    • Protein Size
    • Protein Shape
    • Solution Viscosity
  39. What has Gel Electrophoresis largely been replaced by?
    Paper Chromatography
  40. What are the two most common gels?
    • Polyacrylamide
    • Agarose
  41. Which gel can accommodate molecules of up to 10,000kD?
  42. What is the Maximum size of molecule that can be used with Polyacrylamide Gel?
  43. How do molecules move through an Electrophoresis Gel?
    Through pores in the gel.
  44. How does separation occur in Gel Electrophoresis?
    • Electroporetic Mobility
    • Filtration - Small molecules find it easier to pass through the gel.
  45. What is a Superhydrate?
    A molecular structure capable of filling up with water (and expanding)
  46. With respect to Gel Electrophoresis. Name a Superhydrate.
  47. If a well in an Agarose Gel is filled to 5mm with solution, how big will it's corresponding bands be?
    It's bands will be 5mm thick because band width is equal to well "water" level.
  48. What is the problem with thick bands in Electrophoresis?
    They may overlap, therefore making it impossible to distinguish between the two.
  49. What is Discontinuous Gel Electrophoresis?
    Electrophoresis using different gels of different pH in order to produce narrow bands
  50. What is the basic setup of Discontinuous Gel Electrophoresis?
    • A vertical gel with a pool of buffer solution (pH 8.8-9.0) at the top
    • A Stacking gel (pH 6.8)
    • A resolving gel (pH 8.8)
    • A bottom buffer solution (pH 8.8-9.0)
  51. How are Proteins pushed into Narrow Bands in Discontinuous Gel Electrophoresis?
    The localized charge differences temporarily produced by migrating ions and relatively stationary Glycine Anions mean charges further from the resolving gel move faster than those closer.
  52. Why does the stacking gel have a large pore size?
    Large pores means little size based separation occurs causing all sizes to migrate equally. This allows the bands further back to catch up to the front, regardless of size.
  53. Look!
  54. What is Coomassie Brilliant Blue?
    A protein stain used to detect specific bands.
  55. How does Coomassie Brilliant Blue bind to proteins?
    By denaturing them and binding to their Hydrophobic Core.
  56. Wjat is the Detection Limit of Coomassie Stains?
  57. What is Silver Stain?
    A stain used to detect protein bands in Gel Electrophoresis
  58. What is an Advantage of Silver Stain?
    It is up to 50x more sensitive than Coomassie Brilliant Blue
  59. What are some disadvantages of Coomassie Brilliant Blue?
    • Expensive
    • Difficult to apply
  60. Coomassie Brilliant Blue contains two SO3- groups. Why is it Hydrophobic?
    Because it is mostly made up of [six] non-polar Aromatic rings.
  61. What is an SDS page?
    Electrophoresis separation by size only.
  62. How does an SDS page overcome different charges of proteins?
    A single SDS Detergent molecule binds to a  protein at a ratio of 2 Amino acids for every SDS molecule. This coats the protein in negtive charges
  63. What is the average net charge of 100 Amino Acid Residues in a protein?
    +/- 10
  64. If a typical Polypeptide of 200 residues is coated with SDS what will it's charge be?
    -90 or -110 (Depending on +/- 10 charge on protein)
  65. How does SDS bind to protein?
    By denaturing the protein
  66. What does SDS stand for?
    Sodium Dodecyl Sulphate
  67. How does SDS overcome the problem of different protein folding?
    By denaturing the protein, all proteins are linear
  68. Why is SDS Page alone not effective for proteins with Quaternary Structure?
    The SDS Page will seperate the subunits and they will appear as different proteins
  69. How is the problem of SDS-Page and Quaternary proteins overcome?
    By combining SDS page with SEC
  70. How do you measure the size of proteins from their location in an SDS page?
    Measure their migration and compare it to a standard curve of Relative Mobility bv Log10(Molecular Weight)
  71. Proteins typically contain Disulfide Bridges in their secondary structures. How is this overcome in SDS page?
    Using β-Mercaptoethanol to reduce them.
  72. What is an Assay?
    A method of detecting the presence of a specific molecule quantitatively using another molecule as a specific effector of substrate
  73. What is a Functional Assay?
    An assay which relies on the function (and structure) of a molecule to characterize it.
  74. What are some examples of functional assays?
    • Enzymatic
    • Specific Binding
    • Observable Biological Activity
    • Immunochemistry
  75. In some assays a two step process is used. What are the two steps?
    • Binding of the Molecule (Assay)
    • Detection of the formed complex