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1) Biochemistry I > Protein Purification > Flashcards

Protein Purification Flashcards

1
Q

What is the function of protein purification?

A

Selectively isolating a single protein to analyze it

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2
Q

What should we take advantage of during protein purification?

A

Physiochemical properties

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3
Q

What are the four parameters to look out for during protein purification?

A

1) Solubility
2) Size
3) Charge
4) Affinity Chromatography

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4
Q

What do we hope to achieve through protein purification processes?

A

Augment % of a given protein, while diminishing the % of proteins you’re not interested in

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5
Q

What is the function of the assay method?

A

Allows you to monitor the level of a protein to determine that the desired assay is being enriched

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6
Q

Name the 3 types of assay methods.

A

1) Enzymatic activity
2) Bioassay
3) Immunological-ELISA and immunoblot

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7
Q

Which assay method is highly specific?

A

Immunological-ELISA and immunoblot

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8
Q

How does Immunological-ELISA and immunoblot work?

A

You need to have an antibody that recognizes the protein of interest, amount of colour change = proportional to % (more antibodies recognize)

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9
Q

What does specific activity measure?

A

The purification of a protein from a complex is monitored by specific activity

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10
Q

What is the specific activity formula?

A

Spec. act. = total activity/total protein

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11
Q

What are the units of specific activity?

A

Units of activity/mg of total protein

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12
Q

What does ammonium sulphate precipitation do?

A

Disrupt the water shell that surrounds the protein in solution.

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13
Q

What is a native technique?

A

Techniques that do not denature proteins

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14
Q

Is ammonium sulphate precipitation a native technique?

A

Yes

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15
Q

How do we bring back a protein into a solution after we have performed ammonium sulphate precipitation?

A

Add a buffer

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16
Q

Is ammonium sulphate precipitation powerful? Name an advantage

A

Not powerful, but can use in large amounts

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17
Q

Is there a protocol for protein purification?

A

No protocol, make sure its logical and specific activity goes up

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18
Q

Name the three types of chromatography and their parameters.

A

1) Gel filtration (size)
2) Ion exchange (charge)
3) Ligand or antibodies (affinity)

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19
Q

Define chromatography.

A

The process of differentially separating molecules on the basis of their interaction with a solid support.

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20
Q

What does chromatography require?

A

1) Stationary phase

2) Mobile phase

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21
Q

Define the stationary phase. Give examples.

A

Support matrix, with which components to be separated interact
ex: chalk, TLC

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22
Q

Define the mobile phase. Give examples.

A

Solvent that promotes the differential partitioning of a molecule from the stationary phase.
ex: alcohol, gas

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23
Q

Do compounds move during chromatography?

A

Yes, they jump from the stationary to the mobile phase.

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24
Q

What can we say about compounds that come out first during chromatography?

A

Weaker interaction with the stationary phase

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25
Q

Gel permeation separates based on what parameter?

A

Basis of size

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26
Q

What happens to the small proteins during gel permeation? And the big?

A

Small proteins will diffuse to the beads, spend more time inside
Big proteins will stay on the outside and will come OFF the column first

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27
Q

What type of proteins come out first during gel permeation?

A

Big proteins, since they do not partition through the mbeads

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28
Q

Is gel permeation chromatography a native technique?

A

Yes

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29
Q

Name the 2 types of ion exchange chromatography.

A

1) Cation

2) Anion

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30
Q

Ion exchange separates based on what parameter?

A

Charge

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31
Q

For cation ion exchange, what is the charge on the beads? What will bind?

A

Beads: -
+ proteins will bind
- proteins will repel and shoot out

32
Q

During cation ion exchange, how do we get the bound proteins off? What do we call this process?

A

Adding salt will push the + charged proteins off

sodium ion displacement

33
Q

Explain the concept of mass action and how it relates to ion exchange chromatography.

A

Lower sodium concentration is needed to knock off ++ proteins vs +++++ proteins
So, concentration of sodium will need to increase gradually to knock them all off

34
Q

Which ion would cause displacement during cation ion exchange? Anion?

A

Cation: Na+
Anion: Cl-

35
Q

Is ion exchange chromatography a native technique?

A

In most cases, it does not denature (99%)

36
Q

Why do we use beads for ion exchange chromatography?

A

Minimizing Eddy currents

37
Q

Resume ion exchange chromatography in 3 steps.

A

1) Apply resin
2) Wash resin
3) Elution

38
Q

Define affinity chromatography.

A

Used to isolate a single protein from a complex mixture in a single step

39
Q

How does affinity chromatography work?

A 
Stationary phase (substrate or product ligand)
bound by the protein with a high affinity, other stuff doesn't bind
40
Q

How do you elute affinity chromatography?

A

Taking a soluble form of ligand, putting it on top of the column, displace the protein

41
Q

How can you purify proteins without information on its ligands?

A

Metal Affinity Chromatography

42
Q

What is a substrate?

A

Starting material that binds to an enzyme

43
Q

What is a ligand?

A

Molecule that is bound to a protein. Can be a substrate or a product.

44
Q

Is affinity chromatography a native technique?

A

Usually, yes

45
Q

What is a denaturing technique?

A

Results in the loss of protein activity

46
Q

Is SDS-Page native?

A

No, it is denaturing

47
Q

On what basis does SDS-Page separate on?

A

Size

48
Q

Why do you need to do an SDS-Page every time you do a test

A

Gives you a span of all the molecules contained

49
Q

How do you get 3D gels in electrophoresis?

A

Radical mediated polymerization

50
Q

What pulls the proteins during electrophoresis? According to what?

A

Electric field pulls proteins according to their charge

51
Q

The movement of proteins during electrophoresis depends on what?

A

Size

Small proteins will pass through much faster in the gel than large

52
Q

What is the mobile phase of electrophoresis?

A

Electrical field

53
Q

Why do we want all proteins to be negative during electrophoresis?

A

Because + proteins will get pushed out of the reservoir by + pole

54
Q

What adds the - charge to proteins? What kind of substance is it?

A

SDS, with a sulphate groupe, amphipatic

55
Q

What does the SDS do to the proteins?

A

Binds to and unfolds all the proteins, which will be coated in SDS, and all contain a - charge

56
Q

What is the charge to mass ratio for SDS? What does it mean?

A

Ratio: 1

Bigger proteins will need more SDS

57
Q

What does the rate of movement depend on for SDS page?

A

ONLY depend on size

58
Q

For every g of protein, how many grams of SDS will bind?

A

1.4 g of SDS

59
Q

How do we make proteins linear? Why doesn’t heat work?

A

We need to break disulphide bonds, heat doesn’t work.

B-mercoptoethanol is added and then boiled

60
Q

Why is SDS-Page useful?

A

We can get an accurate measurement of the masses of proteins, using molecular weight ladders

61
Q

What does two-dimensional polyacrylamide separate on the basis of?

A

Charge AND size

62
Q

What makes proteins have different isoelectric points?

A

Depending on the number of acids or bases in the protein

63
Q

What is the first dimension of polyacrylamide gel electrophoresis?

A

Isoelectric point (focusing)

64
Q

How will proteins move during the 1st dimension of polyacrylamide gel electrophoresis?

A

Proteins in the middle that have a - charge will move to + until they reach the pH where the net charge is 0 (pH gradient)

65
Q

What is the 2nd dimension of polyacrylamide gel electrophoresis?

A

Size

66
Q

What happens during the 2nd dimension of polyacrylamide gel electrophoresis?

A

Boil with B-mercoptoethanol and use SDS page

67
Q

How are pH gradients made?

A

Basic side flows first, then acidic will slowly neutralize basic middle, then just acid

68
Q

Why can acids and bases make pH gradients?

A

Because they have different pkas

69
Q

What makes pH gradients form a gel?

A

Acrylamide

70
Q

Acidic proteins will move where during polyacrylamide gel electrophoresis?

A

Acidic (-) will move to +

71
Q

How can we analyze diseases using polyacrylamide gel electrophoresis?

A

Overlaying results

72
Q

Name the technique: taking advantage of unique structural or functional properties of a protein, this technique specifically removes the protein of interest from a solution.

A

Affinity chromatography

73
Q

Name the technique: proteins leave the mobile phase,, associating with a negatively charged immobile substrate, such as a bead or resin.

A

Cation exchange chromatography

74
Q

Name the technique: proteins are separated on the basis of their ability to migrate in an electric field, an indicator of relative size

A

Electrophoresis

75
Q

Name the technique: proteins are chromatographically separated solely on the basis of size

A

Gel filtration

1) Biochemistry I (12 decks)

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