BIC Lecture 4

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  1. Explain the uses of mice in lab settings.

    What did they discover?
    have particular characteristics--> interbree wice with conditions (such as lupus)--> create strains of mice

    researchers who were trying to study mice with lupus noticed that they had particular regenerative properties--> study of mammalian regeneration
  2. Steps to examine a mouse?
    • 1) identify the body part that regenerates
    • 2) euthanize the specimen (dry ice until suffocation)
    • 3) remove/ dissect body parts with charactericts of regeneration
    • 4) isolate
    • 5) characterize
  3. Isolation:

    What is key?
    • along hte way, as we break down the molecules, we need to make sure our protein of interest is still present in the molecule
    • --> throughout isolation, we separate out several molecules. To ensure that our molecule is still present, we can take a small portion of the mixture (aliquot) and see if pyruvate is forming. If so, we continue, If not, we backtrak.
  4. Every isolation has a different __.

    After isolating, what occurs?

    we grind up the molecule in the presence of a buffer--> crude homogenate

    some molecule from the tissue is soluble and goes into the solution. The insoluble solution is what we hope to get rid of. 

    This is done by centrifugation
  5. What is centrifugation?
    solutions are spun at different speeds under different lengths of time. For each round, the pellets form (precipitate out). These pellets are the insoluble parts. 

    Above is the liquid. Buffer + soluble parts= supernatant
  6. Explain what happens depending on whether the protein of interest is in the supernatant or pellet.
    • If in supernatant, it is good
    • If not soluble and in the pellet, difficulty arises, but it is possible
  7. What is the next step after centrifugation?
    Salting out of proteins
  8. Explain salting out of proteins.
    Proteins have different solubilities in water. Certain amounts of water are required to solubilize the protein. So, to separate these proteins from one another, we introduce a highly soluble salt into the solution, such as ammonium sulfate, to cause the proteins to precipitate out one at a time. As AS begins to dissolve, water from the protein requiring the most water is being away by AS to solubilize it. As a result, this protein begins to precipitate, leaving the other two still soluble in solution
  9. Explain further what happens after the first of the several proteins begins to precipitate.
    We centrifuge it to isolate the protein. 

    Afterwards, we repeat the procedure to cause the other proteins to precipitate, one at a time, centrifuge after each run, and isolate
  10. What is this process that you just explained called?
    fractionation: separating out the molecules so that, now, in each test tube, you have less present, but you isolated each
  11. After salting out, what occurs?
    separation by size, the first being dialysis
  12. Explain dialysis
    Proteins are in a buffer. They are all of different sizes. you place it in a dialysis tube with a porous membrane (holes that allow some proteins to travel and others to remain in the dialysis bag
  13. What do you after dialysis has occurred?
    you assay the outside. Assay the inside. Keep going forward with your results. This has led to some separation, but not complete separation.
  14. What information do some dialysis bags contain?
    On the dialysis tube, you will see something that says MWCO to indicate the size that can pass through the bag. Ex: 25 kb--> anything less than 25 kb can get through
  15. After dialysis, what is the next separation technique?
    The separation of molecules by passing molecules in a liquid phase over or through a stationary phase

    Liquid phase: your buffer with your molecule in it
  16. Explain chromatography
    In order to do this, we need more than a bag. 

    Glass tube with filter at the bottom to allow liquid, but nothing else, to get through 

    Top: reservoir with buffer

    Column with beads: polymer beads, which can vary depending on the type of chromatography being performed. There are channels through the beads to allow things to move through. Size varies
  17. What are the phases?
    mobile phase: buffer

    stationary phase: beads with channels
  18. As proteins flow through columns, depending on the size, they will move at __. 

    Explain the relation between the size and the time it takes for one molecule to exit the column.
    different rates

    Largest are the fastest: Because they cannot fit into any pores in the beads, they will move around the beads and have the shortest trip with no detours--> first ones out

    Intermediate: They have a slightly longer pathway due to their ability to fit into some pores, but not all. As a result, they will emerge second.

    Smallest: They are the longest because they can fit through every pore and navigate through all of them.
  19. When the chromatography has finished, what must be done?
    find our proteins (all of them) by determining which tubes actually have proteins in them. Two amino acids (tryptophan and tyrosine) are unique in their resonance (movement of electrons) and can be seen with a spectrophotometer due to their ability to absorb wavelengths at 289 nm.
  20. When we use the spectrophotometer, what do we do? What do we get? What do we do after we get what we get?

    What do we do then?
    We put all of our fractions, one by one, and put them in at 289 nm

    We get a reading with numerous peaks and overrides

    • We check fractions and see where enzyme of interest is located
    • --> throw out fractions that don't have that protein
  21. What is the second chromatography step?
    Separate by charge

    Column full of beads but no channel. They are charged (can be - or +)

    • This is done because your protein carries a strong positive or negative charge
    • -> If positive, get the beads with negative charge
  22. What is this separation by charge called? 

    What are the components?

    buffer with pH set so our protein carries the most charge

    • ionic bonds form and stick to beads
    • Those with weak charges do not bind and pass through
  23. What does the mobile phase enable?
    allows passage through (buffer)
  24. Detecting the protein can be done how? What result should you expect?
    assayed--> not in the solution--> your protein is on the beads
  25. To get them off the beads, what do you do?
    break the ionic bond

    Positively charged at pH of  means there are ionizable groups. So, we get them to a deprotonated state to make them unatttracted to positive charges
  26. What do you do after getting them off the beads?
    assay again
  27. Third type of chromatography 

    What do you do?
    affinity: we have prior knowledge that our protein binds to a certain molecule (for ex: glucose)

    • 1) Make column
    • 2) fill with stationary phase of beads with glucose molecule attached --> column is a trap
    • 3) Proteins wth no affinity flow through, causing separation
  28. In affinity chromatography, where is our protein?
    It is in the column. To get it off, we use a buffer that has solubilized glucose

    Any molecular interaction that involves weak bonds are always letting go and reattaching; changing the elution buffer leads to release of the protein
  29. What is an elution buffer?
    In column chromatography, it is put in the column to basically cleanse and lubricate. Generally, it helps to wash out any left-over proteins from a previous experiment. It can also help to separate the fractions that are collected.
  30. Regarding the chromatography, which molecules can do what?
    all proteins can have size exclusion chromatography done

    Some proteins can undergo Ion-exclusion chromatography

    Few can undergo affinity chromatography
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BIC Lecture 4
Test One
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