biochem 003 techniques in protein biochemistry part 1 (proteome protein purification chromatograph

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biochem 003 techniques in protein biochemistry part 1 (proteome protein purification chromatograph
2014-10-04 18:43:03
biochem 003 techniques protein biochemistry part proteome purification chromatography SDS PAGE isoelectric focusing ELISA
biochem 003 techniques in protein biochemistry part 1 (proteome, protein purification, chromatography, SDS PAGE, isoelectric focusing, ELISA) #5
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  1. what is the proteome>
    the proteome is the entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions
  2. how are enzyme purifications monitored?
    by determining the specific activity of the enzyme being purified.
  3. what is specific activity?
    the ratio of enzyme activity to protein concerntration
  4. with each purification step, what should happen to the specific activity of the enzyme that you are purifying? why?
    it should increase, because, over time, the enzyme of interest comprises a larger part of the total protein concentration
  5. cells are disrupted to form ________
  6. when forming cell homogenate, what should one do to prevent one's protein of interest from being destroyed by proteases?
    use protease inhibitors when forming the cell homogenate
  7. what is the first pellet formed from the centrifugation of cell homogenate?
    nuclear fraction
  8. what is the second pellet formed from the centrifugation of cell homogenate?
    mitochondrial fraction
  9. what is the third pellet formed from the centrifugation of cell homogenate?
    the microsomal fraction
  10. true or false? proteins require a certain amount of salts in the solvent in order to dissolve in the solvent
  11. what is salting out?
    as the salt concentration is increased, different proteins will precipitate out at different salt concentrations, a process called salting out
  12. how can salts be removed from a protein solution?
    the salt can be removed from a protein solution by dialysis. The protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse, but big enough to allow the salt to equilibrate with the solution surrounding the dialysis bag
  13. what is gel filtration chromatography?
    gel filtration chromatography allows the separation of proteins on the basis of size. A glass column is filled with porous beads. When a protein solution is passed over the beads, large proteins cannot enter the beads and exit the column first. Small proteins can enter the beads and thus have a longer path and exit the column last
  14. what is ion exchange chromatography?
    ion excahnge chromatography allows separation of proteins on the basis of charge. The beads in the column are made so as to have a charge. When a mixture of proteins are passed through the column, proteins with the same charge as on the column will exit the column quickly. Proteins with the opposite charge will bind to the beads, and are subsequently released by increasing the salt concentration or adjusting the pH of the buffer that is passed through the column
  15. what is affinity chromatography?
    affinity chromatography takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups. Beads are made with the speciic chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical to which the protein is bound
  16. what is the purpose of SDS in the SDS-PAGE?
    the purpose of SDS is to denature proteins. It also binds to proteins-- 1 molecule of SDS for every 2 amino acids-- giving all the proteins being run in the gel the same charge-to-mass ratio.
  17. all of the proteins run in an SDS PAGE have a ______ charge and migrate to the ______ end of the gel
    negative, positive
  18. What type of protein reaches the positive end of the SDS-PAGE gel first?
    the proteins of smaller mass reach the positive end first
  19. what is isoelectric focusing?
    isoelectric focusing allws the separation of proteins in a gel on the basis of their relative amounts of acidic and basic amino acids. If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate to their isoelectric point, the pH at which they have no net charge
  20. To identifyt the estradiol receptor, the protein mixture from a rat uterus is first incubated with radioactive ______
    radioactive estradiol