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Structure and Function of Biomolecules > Protein techniques > Flashcards

Protein techniques Flashcards

1
Q

What is circular dichroism?

A

A technique that involves shooting a circularly polarized beam of light at a protein solution

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2
Q

What does circular dichroism tell us?

A

The content of secondary structures - how many helices, sheets, and how much random coiling

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3
Q

What is the first step in protein purification?

A

Disrupt the cells. Get them out of the cells and into a solution where we can actually do something with them

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4
Q

What is the resulting solution called after disrupting the cells? What is in it?

A

Crude lysate. Contains proteins and cell debris

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5
Q

How do you isolate all soluble proteins from a crude lysate? What is the new solution called?

A

Centrifuge it to precipitate all the debris into the pellet, then take the supernatant that would have the proteins. Called the clear lysate

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6
Q

What is the next step to start isolating a protein of interest from the cleared lysate?

A

Ammonium sulfate cut

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7
Q

What difference between proteins does an ammonium sulfate cut exploit?

A

Protein solubility

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8
Q

Why does ammonium sulfate allow us to separate proteins?

A

It makes them less soluble, so less soluble proteins will fall out of solution and the cleared lysate will have fewer proteins in it

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9
Q

How do we determine how much ammonium sulfate to add while doing an ammonium sulfate cut?

A

Needs to be determined experimentally

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10
Q

What salts will make proteins more soluble?

A

Guanidinium and SCN-

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11
Q

What is the purpose of dialysis?

A

Remove salts from the ammonium sulfate cut or exchange the buffer a protein is in

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12
Q

How does dialysis work?

A

The protein solution is inside a semipermeable membrane, then put into a salt free solvent. The salt will diffuse out and leave the proteins inside

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13
Q

What is chromatography?

A

Separation of mixtures by using their affinities to the stationary and mobile phases

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14
Q

What is the stationary phase?

A

The solid matrix in the column

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15
Q

What is the mobile phase?

A

The liquid that is moving through the column

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16
Q

Is chromatography denaturing or non denaturing?

A

Non denaturing

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17
Q

What property does ion exchange chromatography separate proteins based on?

A

Charge

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18
Q

What is the stationary phase in ion exchange chromatography?

A

Charged resin

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19
Q

What type of resin would be used to separate out proteins with a positive charge?

A

Cation exchange, would use negatively charged resin that has sulfate anions attached to the resin

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20
Q

What type of resin would be used to separate out proteins with a negative charge?

A

Anion exchange would use positively charged resin that has quaternary amines attached to the resin

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21
Q

Since most proteins aren’t coloured, how do we detect when proteins are being eluted out?

A

A280 absorption. Shine a UV light through the solution as it is coming out and absorption will be registered by a detector

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22
Q

How does the pH affect ion exchange chromatography?

A

We can change the pH to make our protein of interest into a cation or anion

23
Q

What is the mobile phase in ion exchange chromatography?

A

Solution of increasing salt concentrations

24
Q

Why do we increase the salt concentration in the mobile phase for ion exchange chromatography?

A

Increasing the salt disrupts the ionic interactions with the stationary phase, so less charged proteins will let go and get eluted, while more charged will stay stuck

25
What type of separation is being used when the solute concentration in the mobile phase is either increasing or decreasing?
Gradient separation
26
What property does hydrophobic interaction chromatography separate proteins on?
Hydrophobic interactions
27
What is the mobile phase in hydrophobic interaction chromatography?
Solution of decreasing salt concentrations
28
Why does the salt concentration decrease for the mobile phase of hydrophobic interaction chromatography?
Starting the environment out very polar will encourage hydrophobic interactions, and decreasing the salt after that will cause the weaker ones to let go
29
What property does size exclusion chromatography separate proteins on?
Protein size
30
What is the stationary phase in size exclusion chromatography?
Resin beads with small pores in them
31
Do larger or smaller proteins elute faster in size exclusion chromatography?
Larger. They don't go into the pores and get stuck there
32
What property does affinity chromatography separate proteins on?
Interactions with a certain ligand
33
What is the stationary phase in affinity chromatography?
Resin beads with the ligand attached
34
Why is affinity chromatography so effective?
Only the protein of interest will stick to the resin, so everything else will be in the flow through and the protein of interest will be in the column
35
What are 3 ways to elute our protein of interest off the stationary phase in affinity chromatography?
Add solution with soluble ligand, decrease the affinity between the protein and the ligand, or cleave the tag off the protein while in the column
36
How can protein/ligand affinity be decreased to get a protein off the stationary phase in affinity chromatography?
Change the pH
37
How can we cleave an affinity tag off a protein still in the column?
Prescission protease, will cut a very specific sequence that we can engineer into our protein
38
Why do we need to use affinity tags in affinity chromatography?
So the protein will stick to the column when it doesn't have a ligand
39
What are 4 common affinity tags used for affinity chromatography?
Histidine tag, GST, MBP, streptavidin
40
What type of resin does a histidine tag interact with?
Nickel 2+ resin
41
How do you elute a protein with a histidine tag off the column?
Add soluble imidazole (the histidine side group)
42
What type of resin does a GST tag bind to?
Glutathione resin
43
What type of resin does an MBP tag bind to?
Maltose resin
44
What type of resin does a strepavidin tag bind to?
Biotin resin
45
What is a pull down experiment?
Add a protein mixture to some resin beads containing a suspected binding partner, and it will stick to the beads if it binds to that binding partner and we can separate it out
46
What are the two instruments to do chromatography faster than gravity?
HPLC and FPLC
47
How can protein activity be used to purify proteins?
Useful for separating enzymes, use the reaction rate to determine total activity vs specific activity
48
How will total and specific activity change during purification of an enzyme?
Total activity decreases because there's less overall protein, but specific activity will increase because there is less contamination
49
Why is it required to stain protein gels?
Proteins are usually colourless
50
What are the two non-specific protein gel stains?
Coomassie blue and silver staining (AgNO3)
51
When would be good to use silver staining over coomassie?
It is a lot more sensitive, but also more expensive and has waste disposal problems
52
When would you use an SDS PAGE gel?
To determine the size and estimate the molecular weight of a protein of interest
53
How could you tell with gel electrophoresis how many subunits a protein with quaternary structure has, and if they are linked with disulfide bonds?
With no disulfide bonds, would get a band for each subunit With a disulfide and no reducing agent, would get one band that is the size of every subunit added together With a disulfide and a reducing agent, would get a band for each subunit

Structure and Function of Biomolecules (33 decks)

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