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Core Lectures > Protein Purification and Analysis > Flashcards

Protein Purification and Analysis Flashcards

1
Q

What are the 4 levels of protein structure?

A

Primary, secondary, tertiary, quaternary

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2
Q

Define primary structure of a protein

A

Linear amino acid sequence of the polypeptide

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3
Q

Define secondary structure of a protein

A

Local structure of linear segments of the polypeptide backbone atoms

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4
Q

Define tertiary structure of a protein

A

Three-dimensional arrangement of all atoms

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5
Q

Define quaternary structure of a protein

A

Arrangement of separate polypeptide chains into the functional protein

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6
Q

Why do we purify proteins?

A

To understand…
Which proteins are involved in the process?
What does each protein do?
How does it do it?
Can we interfere with its function (therapeutics)?
Because functional studies are difficult in cells

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7
Q

To study a protein in vitro, what do we need large amounts of?

A 
Natural sources (tissues, bacteria, plants)
Recombinant protein expression (through cloning)
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8
Q

Which organisms can we clone proteins into to obtain overexpressed proteins?

A

Bacteria (E. coli)
Insect cells (Baculovirus)
Mammalian cells
Yeast

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9
Q

In basic terms, how do we purify a protein?

A

Kill the organism, lyse the cells, purify the protein

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10
Q

What key molecule has been investigated using pure protein?

A

The making of ATP synthase

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11
Q

Does colocalisation of proteins mean interaction?

A

Not necessarily

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12
Q

What methods can we use to look at the structural biology of proteins?

A

X-ray crystallography

Nuclear magnetic resonance (NMR)

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13
Q

What can we do with pure proteins?

A

Look at the structural biology

Carry out binding/enzymatic/functional assays

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14
Q

What is mass spectrometry?

A

A very accurate method of molecular weight determination and identification of proteins

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15
Q

What does a Western blot require?

A

An antibody specific for the protein of interest or for the affinity tag

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16
Q

Briefly outline the steps used to carry out a Western blot analysis

A
  1. Run an SDS-PAGE gel
  2. Transfer to nitrocellulose
  3. Incubate with specific antibodies
  4. Autoradiography
  5. Western blot
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17
Q

What is SDS?

A

A detergent which binds proteins non-specifically

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18
Q

What does SDS stand for?

A

Sodium dodecyl sulphate

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19
Q

What does SDS do to proteins?

A

It linearises them and coats them in a negative charge

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20
Q

What does PAGE stand for?

A

Polyacrylamide gel electrophoresis

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21
Q

Why is it necessary for the SDS-PAGE step when purifying proteins?

A

To separate proteins by their mass

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22
Q

What are absorbance measures for detecting proteins useful for?

A

Quickly screening which purification fraction contains proteins

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23
Q

List some direct ways of detecting a protein of interest in a mixture

A

Absorbance
SDS-PAGE
Western blot
Mass spectrometry

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24
Q

List some indirect ways of detecting a protein of interest in a mixture

A

Activity assay

Using specific properties of the protein

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25
Q

What is GST?

A

A protein of 25kDa that binds Glutathione with high affinity

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26
Q

How do you purify proteins using GST affinity chromatography?

A

Elute protein with a gradient of free reduced Glutathione which competes with immobilised Glutathione for binding GST

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27
Q

What is FLAG?

A

A small peptide that binds specifically to Flag antibody with high affinity

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28
Q

How do you purify proteins using FLAG affinity chromatography?

A

Elute protein containing a Flag tag with free FLAG peptide

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29
Q

Many different types of affinity chromatography tags have been developed and are commercially available, give examples of the most common ones and what they bind to

A

Hexa-histidine (His6) binds to nickel-NTA
GST binds to glutathione
FLAG binds to an anti-Flag antibody

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30
Q

How do you purify proteins using hexa-histidine affinity chromatography?

A

Elute protein with a gradient of Imidazole which competes with histidine for Ni2+ binding

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31
Q

What is the matrix made up of in hexa-histidine affinity chromatography?

A

Ni-NTA agarose

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32
Q

What do histidine amino acids have a strong affinity for?

A

Ni2+

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33
Q

Nowadays, how are the majority of proteins expressed?

A

Recombinantly

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34
Q

What is the most common method of protein purification considering that the majority of proteins are expressed recombinantly?

A

Affinity chromatography

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35
Q

What does the level of purity achieved from affinity chromatography depend on?

A

The tag and protein of interest

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36
Q

It is often necessary to do a 2nd purification step when purifying proteins using affinity chromatography, give examples of these 2nd step purification methods

A

Ion exchange chromatography

Size exclusion chromatography

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37
Q

What method is only possible when using recombinant protein expression?

A

Affinity chromatography

38
Q

What do many vectors contain that is useful for affinity chromatography?

A

An affinity tag

39
Q

How is the protein released in hydrophobic interaction chromatography (HIC)?

A

By lowering the ionic strength

40
Q

What does the matrix in HIC consist of?

A

Beads of hydrophobic polymers

41
Q

How are proteins separated in HIC?

A

By their hydrophobicity (solubility)

42
Q

In HIC, what do the hydrophobic polymer beads bind?

A

Hydrophobic amino acids of proteins at high ionic strength

43
Q

What is the purification of proteins based on?

A

The physical properties of them, e.g. molecular weight, isoelectric point, solubility

44
Q

One purification methods is sufficient to obtain a pure sample of protein, true or false?

A

False, generally need to combine many purification methods such as ion exchange and size exclusion chromatography

45
Q

What does a protein’s hydrophobicity profile depend on?

A

The surrounding salt concentration

46
Q

Many amino acids are hydrophobic, but where do these amino acids tend to be located within the protein structure?

A

On the inside

47
Q

What is the range of molecular weight of proteins?

A

From 2kDa up to 3.8 million Da

48
Q

How is molecular weight expressed?

A

In Dalton (Da)

49
Q

What does a protein’s molecular weight depend on?

A

The number of atoms (amino acids)

50
Q

What is 1Da equal to?

A

~1g/mol

~1.66 E-27 kg

51
Q

At a protein’s isoelectric point, what is the number of positive charges equal to?

A

The number of negative charges

52
Q

At pH values below the isoelectric point, is the protein positive or negatively charged?

A

Positively charged

53
Q

Define isoelectric point (pI)?

A

The pH at which the protein’s net charge is 0

54
Q

What is the charge on an amino acid at neutral pH?

A

Some amino acids are negatively charged, some are positively charged

55
Q

What do the physical properties of proteins depend on?

A

The primary sequence

56
Q

What does the choice of a protein expression system depend on?

A

The properties of the target protein

The type of analysis post-purification

57
Q

What do genes encode?

A

Proteins

58
Q

What are proteins crucial for?

A

Cellular functions

59
Q

How many genes do humans have?

A

~22,000

60
Q

How many proteins do humans have?

A

~100,000

61
Q

Define polypeptides

A

Linear chains of amino acids which fold into a specific structure depending on the sequence

62
Q

Is the backbone of an amino acid common or different in every amino acid?

A

Common to all amino acids

63
Q

Is the side chain of an amino acid common or different in every amino acid?

A

Different in every amino acid

64
Q

What is protein structure and function defined by?

A

Amino acid properties

65
Q

What types of properties can amino acids have?

A

Hydrophobic/hydrophilic
Charged/uncharged
Small/large

66
Q

Large molecules in size exclusion chromatography cannot enter the pores in the matrix so what happens to them?

A

They move rapidly through the column

67
Q

Small molecules in size exclusion chromatography can enter the pores in the matrix, how does this reflect the time these molecules to take to move through the column?

A

They take longer

68
Q

What is size exclusion chromatography otherwise known as?

A

Molecular sieve or gel filtration chromatography

69
Q

What is the matrix of size exclusion chromatography made up of?

A

Porous beads of carbohydrate polymer, e.g. dextran, sepharose or agarose

70
Q

What is the matrix of ion exchange chromatography made up of?

A

Beads of polymers with a charged functional group

71
Q

How does size exclusion chromatography separate proteins?

A

By their size (molecular weight)

72
Q

How does ion exchange chromatography separate proteins?

A

By isoelectric point

73
Q

What charge are anion exchange resins in ion exchange chromatography and what do they bind?

A

Positive and bind negatively charged proteins

74
Q

What charge are cation exchange resins in ion exchange chromatography and what do they bind?

A

Negative and bind positively charged proteins

75
Q

Simply, how does ion exchange chromatography work?

A

The column binds proteins with the opposite charge

76
Q

How are proteins purified in ion exchange chromatography?

A

Salt competes for ion binding sites and allow proteins to be released
Typically use a gradient, e.g. 0 to 1M NaCl

77
Q

In ion exchange chromatography, what happens the greater the charge on a protein?

A

The tighter it binds the resin, the more salt is needed to dislodge it and the later it elutes

78
Q

The charge on a protein is affected by pH, and so what protein purification method is very sensitive to pH?

A

ion exchange chromatography

79
Q

What does the choice of protein purification method depend on?

A

The protein of interest: size, charges, hydrophobicity

80
Q

Before starting protein purification for analysis, what is it important to analyse first and why?

A

The amino acid sequence of the protein to help design an optimal purification stategy

81
Q

What is the maximum absorbance value of the tryptophan chemical group?

A

~280nm

82
Q

What is the maximum absorbance value of the phenylalanine chemical group?

A

~260nm

83
Q

What is the maximum absorbance value of the tyrosine chemical group?

A

~260nm

84
Q

What is the maximum absorbance value of the peptide bond chemical group?

A

~210nm

85
Q

How is absorbance used to detect proteins?

A

As a general method to use the UV absorbing properties of proteins

86
Q

At what wavelength do most proteins absorb at and what is the disadvantage to this?

A

280nm

Doesn’t allow the discrimination between your protein of interest and the contaminants

87
Q

Some proteins have activities or properties that can be analysed in the test tube (in vitro), true or false?

A

True

88
Q

Do proteins have cellular functions?

A

Yes

89
Q

Anything that the protein of interest does that distinguishes it from others can be used for what?

A

An assay, i.e. a method to detect its presence

90
Q

Give examples of indirect methods for detecting proteins

A

Enzymatic activity

Detecting metalloproteins by using the properties of the metals

91
Q

When using pure proteins, you can test the direct interaction between 2 proteins using binding assays, name 2 of examples of these types of assay

A

GST pulldown

Biophysical methods

92
Q

Give an example of a functional assay

A

Phosphorylation assay

Core Lectures (13 decks)

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