Biochem test one (protein purification)

  1. What was the very first protein purification process based on?
    Protein solubility
  2. In order for the protein to be soluble it must_______
    inquire a sphere of hydration
  3. How do you exploit the protein solubility for protein purification?
    Because the different proteins have different solubilities, so you can separate them by this.
  4. Why do we use salt precipitation?
    Because the salt has a stronger attachment to water than protein.
  5. What makes water attracted to salt?
    It is soluble and has a high charge density
  6. what are three salts that can be used for this process
    • Ammonium sulfate
    • sodium chloride
    • Potassium chloride
  7. Describe the basic process of Salt precipitation
    • Take a protein mixture and add around 10% ammonium sulfate
    • Spin it, take the supernatant, add 20% ammonium sulfate
    • repeat for 30,40 so on. the protein will be in one of these tubes
  8. what is the least soluble protein?
  9. what comes out at a strength of three?
  10. what is the most soluble protein? Why?
    Myoglobin, because it's not a normal component of blood it is generally a muscle protein.
  11. what talks the most ammonium sulfate to bring down?
  12. Salt precipitation uses _________ volumes
  13. To replace water with organic solvent it must________
    be miscible with water
  14. alcohol and acetone are________
    good organic solvents to replace water
  15. Describe the basic process of replacing the water with organic solvent
    • add 10% of organic solvent
    • Spin-take supernatant
    • you separate the proteins out and then you aciate them to determine where the protein lays.
  16. what is the danger when using organic solvents?
    it could denature your protein.
  17. why would the organic solvent denature the protein?
    because the inside of the proteins stay together because they are hydrophobic if there is no water around they could start unfolding due to the less hostile environment.
  18. What is the advantage of the organic solvents?
    It can extract out lipids.
  19. What property(s) does Polyethyianglycal (PEG) have?
    hydrophobic and hydrophilic
  20. Why does a protein interact with water?
    Because of the charge
  21. If you remove water from a protein you will remove________
    the charge
  22. When you reach the net charge of protein being zero, what is this known as?
    isoelectric point or (PI)
  23. when is a protein least soluble?
    at the isoelectric point (PI)
  24. What s cenate precipitation?
    when you adjust the pH in discrete units until it leads to precipitation.
  25. this process will not increase purity by a tremendous amount, but it is a first step and it is useful.
    cenate precipitation
  26. Describe, in general, what chromatography is
    It's when you have a matrix of some kind and you apply the protein mixture and the proteins will move down the matrix depending on size/shape
  27. the induction for protein to migrate
    compelling force
  28. the force against movement of the protein is_____
    retarding force
  29. what are some examples of a compelling force?
    • a flow of water over the matrix
    • putting a charge across the matrix
  30. Examples of Retarding forces
    • friction
    • protein may have a specially affinity for the matrix
  31. Paper, Glass coated with silica gel or a column are examples of what?
    different types of chromatography matrix's
  32. the greater the net force the ______the movement
  33. What is the set-up of Thin-layer chromatography?
    • thin sheet of glass that is coated w/ hydrophlic compound.
    • put compound near the bottom and place it into a tank with a beaker saturated with organic solvents.
    • seal tank, flip over the beaker and fill bottom of tank
  34. the solution will began to cliumb the glass in thin-layer chromatography by a process known as________
    capillary action
  35. what are the two layers or phases of thin-layer chromatography?
    • Stationary phase- one silica gel
    • mobile phase- creeping up the plate
  36. The stationary phase is hydro_____ and the mobile phase is hydro____
    • Hydrophilic
    • Hydrophobic
Card Set
Biochem test one (protein purification)