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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
Q

What type of separation is being used when the solute concentration in the mobile phase is either increasing or decreasing?

A

Gradient separation

26
Q

What property does hydrophobic interaction chromatography separate proteins on?

A

Hydrophobic interactions

27
Q

What is the mobile phase in hydrophobic interaction chromatography?

A

Solution of decreasing salt concentrations

28
Q

Why does the salt concentration decrease for the mobile phase of hydrophobic interaction chromatography?

A

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
Q

What property does size exclusion chromatography separate proteins on?

A

Protein size

30
Q

What is the stationary phase in size exclusion chromatography?

A

Resin beads with small pores in them

31
Q

Do larger or smaller proteins elute faster in size exclusion chromatography?

A

Larger. They don’t go into the pores and get stuck there

32
Q

What property does affinity chromatography separate proteins on?

A

Interactions with a certain ligand

33
Q

What is the stationary phase in affinity chromatography?

A

Resin beads with the ligand attached

34
Q

Why is affinity chromatography so effective?

A

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
Q

What are 3 ways to elute our protein of interest off the stationary phase in affinity chromatography?

A

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
Q

How can protein/ligand affinity be decreased to get a protein off the stationary phase in affinity chromatography?

A

Change the pH

37
Q

How can we cleave an affinity tag off a protein still in the column?

A

Prescission protease, will cut a very specific sequence that we can engineer into our protein

38
Q

Why do we need to use affinity tags in affinity chromatography?

A

So the protein will stick to the column when it doesn’t have a ligand

39
Q

What are 4 common affinity tags used for affinity chromatography?

A

Histidine tag, GST, MBP, streptavidin

40
Q

What type of resin does a histidine tag interact with?

A

Nickel 2+ resin

41
Q

How do you elute a protein with a histidine tag off the column?

A

Add soluble imidazole (the histidine side group)

42
Q

What type of resin does a GST tag bind to?

A

Glutathione resin

43
Q

What type of resin does an MBP tag bind to?

A

Maltose resin

44
Q

What type of resin does a strepavidin tag bind to?

A

Biotin resin

45
Q

What is a pull down experiment?

A

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
Q

What are the two instruments to do chromatography faster than gravity?

A

HPLC and FPLC

47
Q

How can protein activity be used to purify proteins?

A

Useful for separating enzymes, use the reaction rate to determine total activity vs specific activity

48
Q

How will total and specific activity change during purification of an enzyme?

A

Total activity decreases because there’s less overall protein, but specific activity will increase because there is less contamination

49
Q

Why is it required to stain protein gels?

A

Proteins are usually colourless

50
Q

What are the two non-specific protein gel stains?

A

Coomassie blue and silver staining (AgNO3)

51
Q

When would be good to use silver staining over coomassie?

A

It is a lot more sensitive, but also more expensive and has waste disposal problems

52
Q

When would you use an SDS PAGE gel?

A

To determine the size and estimate the molecular weight of a protein of interest

53
Q

How could you tell with gel electrophoresis how many subunits a protein with quaternary structure has, and if they are linked with disulfide bonds?

A

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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