The flashcards below were created by user
on FreezingBlue Flashcards.
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
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
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.
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
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
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
What is the next step after centrifugation?
Salting out of proteins
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
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
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
After salting out, what occurs?
separation by size, the first being 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
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.
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
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
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
What are the phases?
mobile phase: buffer
stationary phase: beads with channels
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.
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.
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.
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
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
What is this separation by charge called?
What are the components?
ION EXCHANGE CHROMATOGRAPHY
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
What does the mobile phase enable?
allows passage through (buffer)
Detecting the protein can be done how? What result should you expect?
assayed--> not in the solution--> your protein is on the beads
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
What do you do after getting them off the beads?
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
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
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.
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