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1) Biochemistry I > Protein Purification > Flashcards

Protein Purification Flashcards

(75 cards)

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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
Gel permeation separates based on what parameter?
Basis of size
26
What happens to the small proteins during gel permeation? And the big?
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
27
What type of proteins come out first during gel permeation?
Big proteins, since they do not partition through the mbeads
28
Is gel permeation chromatography a native technique?
Yes
29
Name the 2 types of ion exchange chromatography.
1) Cation | 2) Anion
30
Ion exchange separates based on what parameter?
Charge
31
For cation ion exchange, what is the charge on the beads? What will bind?
Beads: - + proteins will bind - proteins will repel and shoot out
32
During cation ion exchange, how do we get the bound proteins off? What do we call this process?
Adding salt will push the + charged proteins off | sodium ion displacement
33
Explain the concept of mass action and how it relates to ion exchange chromatography.
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
Which ion would cause displacement during cation ion exchange? Anion?
Cation: Na+ Anion: Cl-
35
Is ion exchange chromatography a native technique?
In most cases, it does not denature (99%)
36
Why do we use beads for ion exchange chromatography?
Minimizing Eddy currents
37
Resume ion exchange chromatography in 3 steps.
1) Apply resin 2) Wash resin 3) Elution
38
Define affinity chromatography.
Used to isolate a single protein from a complex mixture in a single step
39
How does affinity chromatography work?
``` Stationary phase (substrate or product ligand) bound by the protein with a high affinity, other stuff doesn't bind ```
40
How do you elute affinity chromatography?
Taking a soluble form of ligand, putting it on top of the column, displace the protein
41
How can you purify proteins without information on its ligands?
Metal Affinity Chromatography
42
What is a substrate?
Starting material that binds to an enzyme
43
What is a ligand?
Molecule that is bound to a protein. Can be a substrate or a product.
44
Is affinity chromatography a native technique?
Usually, yes
45
What is a denaturing technique?
Results in the loss of protein activity
46
Is SDS-Page native?
No, it is denaturing
47
On what basis does SDS-Page separate on?
Size
48
Why do you need to do an SDS-Page every time you do a test
Gives you a span of all the molecules contained
49
How do you get 3D gels in electrophoresis?
Radical mediated polymerization
50
What pulls the proteins during electrophoresis? According to what?
Electric field pulls proteins according to their charge
51
The movement of proteins during electrophoresis depends on what?
Size | Small proteins will pass through much faster in the gel than large
52
What is the mobile phase of electrophoresis?
Electrical field
53
Why do we want all proteins to be negative during electrophoresis?
Because + proteins will get pushed out of the reservoir by + pole
54
What adds the - charge to proteins? What kind of substance is it?
SDS, with a sulphate groupe, amphipatic
55
What does the SDS do to the proteins?
Binds to and unfolds all the proteins, which will be coated in SDS, and all contain a - charge
56
What is the charge to mass ratio for SDS? What does it mean?
Ratio: 1 | Bigger proteins will need more SDS
57
What does the rate of movement depend on for SDS page?
ONLY depend on size
58
For every g of protein, how many grams of SDS will bind?
1.4 g of SDS
59
How do we make proteins linear? Why doesn't heat work?
We need to break disulphide bonds, heat doesn't work. | B-mercoptoethanol is added and then boiled
60
Why is SDS-Page useful?
We can get an accurate measurement of the masses of proteins, using molecular weight ladders
61
What does two-dimensional polyacrylamide separate on the basis of?
Charge AND size
62
What makes proteins have different isoelectric points?
Depending on the number of acids or bases in the protein
63
What is the first dimension of polyacrylamide gel electrophoresis?
Isoelectric point (focusing)
64
How will proteins move during the 1st dimension of polyacrylamide gel electrophoresis?
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
What is the 2nd dimension of polyacrylamide gel electrophoresis?
Size
66
What happens during the 2nd dimension of polyacrylamide gel electrophoresis?
Boil with B-mercoptoethanol and use SDS page
67
How are pH gradients made?
Basic side flows first, then acidic will slowly neutralize basic middle, then just acid
68
Why can acids and bases make pH gradients?
Because they have different pkas
69
What makes pH gradients form a gel?
Acrylamide
70
Acidic proteins will move where during polyacrylamide gel electrophoresis?
Acidic (-) will move to +
71
How can we analyze diseases using polyacrylamide gel electrophoresis?
Overlaying results
72
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.
Affinity chromatography
73
Name the technique: proteins leave the mobile phase,, associating with a negatively charged immobile substrate, such as a bead or resin.
Cation exchange chromatography
74
Name the technique: proteins are separated on the basis of their ability to migrate in an electric field, an indicator of relative size
Electrophoresis
75
Name the technique: proteins are chromatographically separated solely on the basis of size
Gel filtration

1) Biochemistry I (12 decks)

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