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Genomic and Proteomics > Chapter 2 Introduction to protein purification > Flashcards

Chapter 2 Introduction to protein purification Flashcards

1
Q

Objectives

A

Describe the process of cell breakage and protein extraction.

Describe the general principles, strategies and techniques involved in the purification of proteins.

Describe methods used in the assay of proteins.

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2
Q

Under sample preparation between the workflow of sample and protein mixture

A

Sample separation and visualization
Comparative analysis
Digestion

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3
Q

Why does a sample need to be prepared

A
  • solubilize proteins from source into a stable, liquid form for subsequent procedures
  • remove contaminants or unwanted proteins that might affect resolution during visualization e.g (SDS-PAGE)
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4
Q

Cell Disruption

A

Depends on the type of materials from which to extract proteins

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5
Q

3 types of cell disruption

A

Serum, plasma, urine = liquid samples, can be used directly with little pre-treatment

Cells = need for lysis

Tissues = need for homogenisation through mechanical means before lysis
(e.g. plant tissues, fibrous tumours etc.)

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6
Q

Methods of physical lysis / disruption

A
  1. Sonication

Cell suspensions samples subjected to sonication in short bursts to avoid heating and foaming.

High frequency sound waves generated by sonication creates shear forces that lyse cells.

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7
Q

methods of physical lysis / manual grinding

A
  1. Manual grinding

Grinding by mortar and pestle- tissue or cells often frozen in liquid nitrogen and ground to fine powder

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8
Q

methods of physical lysis / mechanical disruption

A
  1. Mechanical disruption

Hand held devices e.g. Dounce homogenizer or blenders can be used to disrupt cell suspensions

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9
Q

5 methods of physical lysis

A

sonication, manual grinding, mechanical disruption, liquid homogenization, rapid freeze thawing

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10
Q

methods of physical lysis / liquid homogenization

A

Cells are lysed by shear forces resulting from forcing the cells suspension through
a small opening under high pressure in a French pressure cell.

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11
Q

methods of physical lysis / rapid freeze thawing

A

Freezing creates ice crystal formation that disrupts cell membranes and if followed by rapid thawing, the cells burst.

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12
Q

2 methods of enzyme or detergent based lysis

A

Enzymatic lysis and detergent lysis

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13
Q

enzymatic lysis

A

Used to digest cell walls of microbes and plants
e.g.
cellulase for plants, 1,3-glucanase for yeast and lysozyme for Gram-negative bacteria.

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14
Q

detergent lysis

A

Uses detergents to disrupt cell membranes

Ionic detergents e.g. SDS (-ve) and CTAB (+ve)

Zwitterionic detergent e.g. CHAPS

Non-ionic detergent e.g. NP-40

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15
Q

Typical lysis protocol for mammalian cells

A
  1. Harvest 5×106 cells and wash with PBS
  2. Add 200µl of lysis buffer
  3. Spin at 14,500 rpm for 1 hr at 4ºC
  4. Store at -80ºC
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16
Q

Composition of typical lysis buffer

A 
Urea (7M)
Thiourea (2M)
CHAPS (4%w/v)
TRIS Base (40mM in water)
DNAse (10mg/ml) 
RNAse (10mg/ml)
PMSF (0.1M)
DTT (1 mM)
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17
Q

chaotropic agents in typical lysis buffer

A

Urea (7M)

Thiourea (2M)

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18
Q

CHAPS (4% w/v)

A

Detergent

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19
Q

TRIS-base (40mM in water)

A

Buffer

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20
Q

DNAse (10mg/ml)

A

Digest DNA/RNA

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21
Q

RNAse (10mg/ml)

A

Digest DNA/RNA

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22
Q

PMSF (0.1M)

A

Protease inhibitor

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23
Q

DTT (1mM)

A

Reductant

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24
Q

chaotropic agents

A

Cosolutes that can disrupt the hydrogen bonding network between water molecules and reduce the stability of the native state of proteins by weakening the hydrophobic effect.

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25
Q

The 3 methods of detergent lysis

A
  • Solubilising membrane proteins and lipids.
  • Controlling protein crystallization.
  • Preventing non-specific binding in affinity purification
    and immunoassay procedures
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26
Q

Additional function of detergent

A

Detergents also promote electrophoresis of soluble proteins (e.g. SDS-PAGE)

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27
Q

Additional function of detergent

A

Able to disperse hydrophobic proteins or hydrophobic parts of proteins.

Detergent/surfactant molecules have both hydrophilic and hydrophobic portions

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28
Q

Detergents for lysis

A

CHAPS (zwitterionic), NP-40 (non-ionic) or Triton-X (non-ionic) are commonly used (compatible with IEF)

SDS (anionic) may be used sparingly for difficult samples

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29
Q

Types of detergent to be used

A

Detergents used must be zwitterionic or nonionic to prevent complications during downstream applications e.g. isoelectric focusing (IEF).

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30
Q

Solubilizing action of detergents

A

before solubilization

after solubilization

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31
Q

Structure of detergents (CHAPS)

A

CHAPS (zwitterion)

There is a hydrophobic and hydrophilic halves of the molecules

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32
Q

Structure of detergents (NP40)

A

NP40 (monionic)

There is a hydrophobic and hydrophilic halves of the molecules

Hydrophobic is C8H17 bound to a benzene

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33
Q

Solubilizing effect of detergents

A

Proteins are held in the lipid bilayer by hydrophobic interactions between the lipid tails and hydrophobic protein domains.

  1. These integral membrane proteins (IMPs) are not soluble in aqueous solutions as they aggregate to protect their hydrophobic domains, but are soluble in detergent solutions as micelles formed by detergents are analogous to the bilayers of the biological membranes.
  2. Proteins are incorporated into these micelles via hydrophobic interactions. Hydrophobic regions of membrane proteins, normally embedded in the membrane lipid bilayer, are now surrounded by a layer of detergent molecules and the hydrophilic regions are exposed to the aqueous medium.
    This keeps the membrane proteins in solution.

Complete removal of detergent could result in aggregation due to the clustering of hydrophobic regions and, hence, may cause precipitation of membrane proteins

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34
Q

Micelle

A

The inside is hydrophobic and outside is hydrophillic

Native membrane -> solubilization and purification

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35
Q

Chaotrope or chaotropic agents in slides

A

A chaotrope disrupts hydrogen bonding and hydrophobic interactions between and within proteins.

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36
Q

Result of addition of chaotropic agents

A

This action breaks proteins and convert proteins from native conformation into a random conformation thereby solubilising them

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37
Q

Why urea is used at 8M

A

Urea used at 8M unfolds most proteins

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38
Q

Why urea is used with thiourea

A

Urea is used together with thiourea to solubilise membrane proteins.

Thiourea increases solubilisation power of urea.

Urea/thiourea has to be prepared fresh to reduce conversion to cyanate/thiocyanate.

Cyanate/thiocyanate can cause carbamylation which changes MW of solubilized proteins.

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39
Q

Carbamylation

A

Carbamylation is a non-enzymatic spontaneous reaction of a primary amine or a free sulfhydryl group of protein with isocyanate

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40
Q

Carbmylation as a problem

A

In 2-D gel analysis and the associated sample preparation steps.

This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point.
It also changes the MW of solubilized proteins

41
Q

How do carbamides form

A

Isocyanic acid reacts withaminesto giveureas(carbamides)

42
Q

Carbamylation

A

HNCO + RNH2→ RNHC(O)NH2

43
Q

Reductants or reducing agents

A

Reducing agents or reductants break apart intramolecular and intermolecular disulphide bonds in proteins and maintain them in fully reduced state (-SH)

44
Q

DTT (dithiothreitol)

A

Commonly used as a reducing agent

45
Q

Reason DTT is preferred over beta-mercaptoethanol

A

DTT is preferred over β-mercaptoethanol because of its water solubility and lower toxicity.

46
Q

Danger with beta-mercaptoethanol

A

2‑Mercaptoethanol is considered toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.

47
Q

Why do we use detergents, chaotropes and reductants

A

Various components of the lysis buffer break apart proteins to denatured forms.

Protein-protein interactions complicate identification.

There is a need to reduce proteins to their smallest unit possible.

48
Q

Small issue with 2D-PAGE

A

Note, some protein complexes are too large to be resolved using 2D-PAGE.

49
Q

Removal of contaminants : DNA/mRNA
Reasons DNA/mRNA is removed
How DNA/mRNA is removed

A

DNA and mRNA are released into lysis buffer.

DNA/mRNA in prepared sample increases viscosity of the soluble protein and makes handling difficult (e.g. pipetting).

Some protein form complexes with DNA/mRNA.

We add nucleases (DNase/Rnase) digests DNA/mRNA

50
Q

Removal of contaminants : cell debris

How cellular membrane fragments are moved

A

cellular membranes remain as fragments after lysis

We remove them with simple step of centrifugation

  • cellular membrane fragments will pellet after centrifugation while soluble proteins remains in the supernatant
51
Q

Typical lysis protocol for mammalian cells

A

Spin at 14.500 rpm for 1 hour at 4 degree Celsius

52
Q

Temperature for storage and handling of samples

A

Store at -80 degree Celsius

53
Q

Typical lysis protocol for mammalian cells

A
  1. Harvest 5×106 cells and wash with PBS
  2. Add 200µl of lysis buffer
  3. Spin at 14,500 rpm for 1 hr at 4ºC
  4. Store at -80ºC
54
Q

Proteases released during cell lysis

A

Proteases are released by cells during lysis, when the lysosomes of cells are compromised

Proteases degrade proteins and cause problems during sample preparation

55
Q

3 methods of handling proteases released during cell lysis

A

Prepare and handle sample at low temperature (e.g. on ice).

Long-term storage at -80ºC or -20ºC.

Add protease inhibitors to preserve quality of proteins used for subsequent steps.

56
Q

Endopeptidases (common proteases)

A

A broad range of enzymes catalyze the hydrolysis of peptide bonds in the interior of a polypeptide chain or protein molecule

57
Q

Mechanism of action by endopeptidases

A

Nucleophilic attack by OH-group on the carbonyl group of the peptide bond

Thus, enabling the cleavage of the peptide bond via hydrolysis

58
Q

5 common protein inhibitors

A 
PMSF 
EGTA/EDTA 
Leupeptin 
Pepstatin 
Aprotinin
59
Q

PMSF

A

Against serine, cysteine proteases

60
Q

EGTA/EDTA

A

Against metalloproteases

61
Q

Leupeptin

A

Against serine/cysteine proteases

62
Q

Pepstatin

A

Against aspartyl proteases

63
Q

Aprotinin

A

Against serine proteases

64
Q

Example of sample preparation protocol by proteomic analysis

A
  1. Protease inhibitors
  2. Freeze thawing
  3. Detergent and reductant
  4. Nucleases
  5. Protease inhibitors, detergent
  6. Chaotropic agents detergent
65
Q

Protein prefractionation

A

Prefractionation may be carried out to enrich for specific proteins

> Studying proteins associated with certain cellular organelles/compartments or studying certain classes of proteins

> Some proteins occur at a low abundance relative to other

Plasma/serum immunoglobulins are abundant and thus need to remove abundant unwanted proteins

66
Q

2 reasons for prefractionation

A
  1. Reducing the amount of unwanted proteins increases the amount of proteins that can be loaded on 2D-PAGE.

This facilitates detection of low abundance proteins.

  • The loading of protein on an IEF gel is limited at a maximum ~120µg)
    2. Reducing protein complexity improves resolution in 2D-PAGE.
67
Q

Use of ion exchange chromatography for prefractionation

A

Separating a sample mixture by applying it through a medium in which different components move at different rates

68
Q

Pre fractionation

A

Prior fractionation of proteins

Try to use a fractionation methods that generates minimal protein overlap between fractions

69
Q

3 common methods of prefractionation

A
  1. immunoaffinity depletion
  2. affinity chromatography
  3. subcellular fractionation
70
Q

Principles of Immunoaffinity depletion

A

A form of affinity chromatography.

Antibodies to “unwanted” proteins are immobilized onto a solid matrix in a column.

Protein samples are applied to the column and “unwanted” proteins bind to the column.

Remaining “wanted” proteins flow through and are collected.

71
Q

Immunoaffinity depletion

A

“Unwanted” proteins trapped and remaining :wanted proteins flow through and collected

72
Q

Affinity chromatography

A

“Wanted proteins trapped and eluted later”

“Unwanted proteins flow through and discarded”

73
Q

Antibody affinity chromatography

A 
1. Load in pH7 buffer
Proteins recognized by antibodies 
Protein not recognized by antibodies
2. Wash
3. Elute with pH 3 buffer
74
Q

Subcellular fractionation

A

To enrich the sample with certain organelles (nuclear or cytosolic fractions, ER, mitochondria) for investigation of certain classes of proteins.

Mechanical homogenisation followed by various steps of centrifugation and/or ultracentrifugation.

75
Q

Process of subcellular fractionation

A

Filter homogenate to remove clumps of unbroken cells, connective tissues etc

Filtered homogenate 600g x 10 min

Nuclei pour out 15,000g x 5 min

Mitochondria, chloroplasts, lysosome and peroxisome pour out 100,000g x 60 minutes

Plasma membrane, microsomal fraction (fragments of endoplasmic reticulum) and large polyribosomes
pour out 300,000g x 2 hours

Ribosomal subunits, small polyribosomes pour out soluble portion of cytoplasm

76
Q

Ultra centrifugation vs centrifugation

A

Using different centrifugal forces allow fractionation of cellular components of various densities

Ultracentrifugation is 80,000g while centrifugation is 800g

77
Q

Why use a mix of ultra centrifugation and centrifugation

A

Using different centrifugal forces allow fractionation of cellular components of various densities

78
Q

5 examples protein quantification and detection methods

A

UV spectrometry
Colorimetric assays

Other methods: (e.g Amino acid analysis, radio-labelling, RP-chromatography)

79
Q

principle of UV-spectrophotometry

A

Absorbance of light at near UV (280 nm) by protein is dependent mainly on tyrosine and tryptophan content and to a lesser extent phenylalanine

80
Q

Advantages of protein determination using UV-Vi spectrophotometry

A
  • Method is simple and fast.

- The sample can be recovered.

81
Q

Disadvantages of protein determination using UV-Vi spectrophotometry

A

Interference from other chromophores.

Specific absorption value for a given protein must be determined.

Presence of contaminant nucleic acid significantly increases absorbance at 280 nm.

82
Q

Examples of principle of protein denaturation using UV-spectrophotometry

A

Cuvette-based spectrophotometer, Nanodrop device

83
Q

Colorimetric assays for protein quantification

A

Quantification of total protein content is required to determine sample loading

84
Q

Commonly employed assays include

A
  1. Bicinchoninic acid assay (BCA)

2. Bradford assay

85
Q

BCA(bicinchoninic acid) assay 2 steps

A

Step 1: Biuret reaction
Protein + Cu+2 -> Cu+1 Reduction fvia OH-
Step 2:
Cu-1 + 2BCA+ -> BCA+ Cu complexe

86
Q

Principles of the 2 steps in bicinchoninic acid

A

Step 1: The BCA assay method involves the reduction of cupric ions to cuprous ions by proteins, in an alkaline reaction (biuret reaction)

Step 2: 2 molecules of bicinchoninic acid chelates with 1 molecule of cuprous ion to form a purple colored complex that absorbs light at 562nm

Note: purple complex is read at 562nm on spectrophotometer

87
Q

Cupric

A

Copper with a valency of two; of copper(II).

88
Q

Cuprous

A

Copper with a valence of one.

89
Q

Bradford assay

A

Protein (basic and aromatic side chains) + Coomassie* G-250 forms a protein dye complex

is blue at 595nm

90
Q

Principles of bradford assay

A

Proteins containing basic or aromatic amino acid side chains bind by hydrophobic or Van der Waal’s interactions with Coomassie Brilliant Blue dye.

This causes a spectral shift from red/brown form of the dye (absorbance at 465 nm) to the blue form of the dye (absorbance at 595nm)

The more blue the bradford assay, the more proteins it has

91
Q

Recap of basic and aromatic amino acids

A

Basic side chains - lysine, histidine, arginine

Aromatic side chains - tyrosine, phenylalanine and tryptophan

92
Q

Substances that interfere with protein colourmetric assay

A

Certain substances react directly with reagents in protein assay, giving “higher” inaccurate readings

Different thresholds for different assays

Choice of assay depends on the composition of sample

93
Q

What is threshold in protein colorimetric assay?

A

Threshold is the amount of the interfering substance if exceeded will interfere with assay

94
Q

Tolerances for detergents and reducing and thiol containing agents of BCA by concentration

A

Brij - 35 5.0%
Brij - 56 1.0%
Brij - 58 1.0%

N-acetylglucosamine in PBS pH7.2 10mM
Ascorbic acid –
Cysteine –

95
Q

Tolerances for detergents and reducing and thiol containing agents of BCA by Coomassie assay

A

Brij - 35 - 0.125%
Brij - 56 - 0.031%
Brij - 58 - 0.031%

N-acetylglucosamine in PBS pH7.2 100mM
Ascorbic acid 50 mM
Cysteine 10 mM

96
Q

Comparison of assays (BCA part)

A

Less variability than bradford

Slower than bradford as it has more steps

Compatible with detergents such as SDS and triton-X

Better detection range (20-2000ug/ml)

97
Q

Comparison of assays (Bradford part)

A

More variability than BCA

Faster than BCA

Compatible with reducing agents

Detection range (1-1,400ug/ml)

98
Q

Know what is the problem with a protocol

A

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Genomic and Proteomics (9 decks)

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