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Analytical and Clinical > Week 2 - Protein Chromatography > Flashcards

Week 2 - Protein Chromatography Flashcards

1
Q

WHAT IS CHROMATOGRAPHY?

A

A technique for analyzing or separating mixtures of gases, liquids or dissolved substances

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2
Q

What are the two stages of chromatography?

A

→ Stationary phase (matrix)

→Mobile phase

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3
Q

CHROMATOGRAPHY–USES

A

Forensics
- blood, arson, post mortem

Food regulation/testing
–horsemeat scandal, nutritional information

Athlete testing
–including horses! (combined liquid chromatography and MS)

Quality control
–alcohol, analysis of when a food spoils, water samples, contaminents

Pharmaceutical industry
–purification of antibodies, creating vaccines, purity of preparations

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4
Q

PRINCIPLES OF CHROMATOGRAPHY–MOBILE AND STATIONARY PHASES

A
  • Mobile phase passed over stationary phase
  • Sample–mobile phase
  • Relative affinity for stationary phase allows for differentiation of sample
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5
Q

Two common methods of chromatography

A

column chromatography, planar chromotography

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6
Q

PRINCIPLES OF CHROMATOGRAPHY–TYPES OF CHROMATOGRAPHY

A
  • Gas Chromatography (GC)
  • Liquid Chromatography (LC)
  • High-Performance Liquid Chromatography (HPLC)
  • Thin-Layer Chromatography (TLC)
  • Other chromatography techniques (exchange chromatography, affinity chromatography)
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7
Q

COLUMN CHROMATOGRAPHY - USES

A
  • Separation of mixtures
  • PurificationIsolation of active components
  • Estimation of drugs in a formulation
  • Isolation of active constituents
  • Separation of diastereomers
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8
Q

COLUMN CHROMATOGRAPHY - VARIABLES

A

Dimension of the column: column efficiency can be improved by increasing length/width ratio of the column.

Particle size of column packing: think sand vs gravel

  • Activity of the adsorbent

Temperature of the column
- speed of the elution increases at higher

Packing of the column

Quality of solvents
- solvents having low viscosities give better results

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9
Q

How does quality of solvents affect chromatography?

A

solvents having low viscosities give better results

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10
Q

How does dimension of the column affect chromatography?

A

column efficiency can be improved by increasing length/width ratio of the column.

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11
Q

PROTEIN EXTRACTION

A

Source of protein
–tissue or microbial cells We need to break open the cells–crude extract

Extraction of proteins
- Homogenization, Sonication, Freeze-thaw cycles, Organic solvents

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12
Q

PROTEIN PURIFICATION–PRELIMINARY STEPS

A

Precipitation and differential solubilization:
- Salting out (Ammonium sulphate)
- Detergents (Triton X-100, CHAPS)–dissolve cell membrane and keep protein in solution

Ultracentrifugation: Sub-cellular organelles

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13
Q

PROTEIN PURIFICATION

A

Need a pure preparation of a protein to determine properties or activity

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14
Q

PROBLEMOF PROTEIN PURIFICATION

A

Cells contain many proteins

  • centrifugation to isolate organelles or fractions
  • Proteins vary in size, charge, binding properties

Genetic engineering–modify proteins but alsofunction

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15
Q

PROTEIN CHROMATOGRAPHY

A

Crude extract fractionation purification of protein of interest..

Fractionation– pH, solubility, temperature …

  • Salting out–depends on lowering solubility with salts e.g. ammonium sulphate
  • Precipitates–centrifugation
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16
Q

PROTEIN CHROMATOGRAPHY - Dialysis

A

remove proteins from small solutes

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17
Q

PROTEIN CHROMATOGRAPHY - Column Chromatography - what does it use to seperate?

A

uses charge, size, binding affinity

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18
Q

PROTEIN CHROMATOGRAPHY - Differences from HPLC/GC

A
  • Low pressure
  • Low temperature
  • Low flow rate
  • Bigger columns/volumes
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19
Q

PROTEIN PURIFICATION - CHROMATOGRAPHY EXAMPLES

A
  • Size-exclusion chromatography
  • Ion-exchange chromatography
  • Affinity chromatography
  • Hydrophobic interaction chromatography
  • Chromatofocussing
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20
Q

What is affinity chromatography based on?

A

Based on binding affinity

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21
Q

How are the beads in the column attached?

A

covalently attached ligand

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22
Q

PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING

A
  • Many proteins do not bind a ligand that can be immobilized on a column
  • BUT gene for almost any protein can be altered to express a fusion protein that can be purified by affinity chromatography
  • Gene encoding the target protein is fused to a gene encoding a peptide or protein that binds a simple, stable ligand with high affinity and specificity–the tag.
  • Tag sequences can be at amino or carboxyl terminus
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23
Q

PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING - EXAMPLES

A

GST tag

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24
Q

GST tag

A

GST enzyme binds to glutathione

Glutathione–immobilised on beads of agarose

  • Retrieve target–wash with high concentration of salt or free glutathione–compete with immobilized ligand for GST binding
  • Possible to remove the tag by protease cleavage
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25
Q

PROTEIN PURIFICATION - METHOD TO CHECK THE PRODUCT PURITY

A

find out the isoelectric point (pI) Approx mw

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26
Q

PROTEIN PURIFICATION - HOW TO CHECK THE PRODUCT PURITY

A

Electrophoresis

–Polyacrylamide gel

– a molecular “sieve”

–slowing migration of proteins in proportion to charge to mass ratio as below

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27
Q

SDS–PAGE

A
  • Common method for estimating purity and mw
  • Uses SDS
  • Binding ratio 1.4x–nearly 1 molecule of SDS to each aa residue
  • Sulfate moiety of bound SDS contributes to net negative charge–intrinsic charge of protein not significant
  • SDS binding partially unfolds proteins–rodlike shapes
  • Separation mainly on basis of mw
  • Visualisation–Coomassie blue binds to proteins
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28
Q

CHROMATOFOCUSSING - OVERVIEW

A
  • Chromatofocusing medium equilibrated with a start buffer at a pH slightly above the highest pH required.
  • Sample is applied to the chromatographic column with the start buffer
  • Elution buffer is passed through the column and begins to titrate the amines on the medium and the proteins–gradient pH develops
  • Proteins in the sample that are at a pH above their pI are negatively charged and retained near the top of the column
  • The ones having their pH below pI begin to migrate down and bind to that part of the column where the pH is above their pI
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29
Q

CHROMATOFOCUSSING

A
  • Binding dependent on the surface charge of the protein
  • Uses ion exchange resins
  • Uses FPLC
  • Elutes bound species by altering the pH of the buffer
  • Proteins elute in order of their isoelectric points.
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30
Q

2D GEL ELECTROPHORESIS - So why not combine SDS page and isoelectric focussing?

A
  • More sensitive than either alone..
  • separates proteins of identical molecular weight that differ in pI, orproteins with similar pI values but different mw
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31
Q

POST CHROMATOGRAPHY PROCESSING

CONCENTRATION

A
  • Lyophilization aka. freeze drying
  • Ultrafiltration
  • Chromatographic concentration
  • Precipitation
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32
Q

POST CHROMATOGRAPHY PROCESSING

YIELD AND ANALYSIS TECHENQUES

A
  • Enzyme assay
  • Protein assay
  • SDS-PAGE
  • Western Blotting
33
Q

POST CHROMATOGRAPHY PROCESSING

YIELD AND ANALYSIS
Simulation

A

SDS-PAGE & immunoblotting

34
Q

POST CHROMATOGRAPHY PROCESSING
RECOMBINANT PROTEINS

A
  • Manipulated form of native protein
  • Generated in various ways in order to increase production of proteins, modify
  • Coding sequence for the protein of interest is isolated and cloned into an expression plasmid vector.
  • Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture.
  • intron-free version of the gene is often made by converting the mRNA into cDNA
  • But cDNA lacks regulatory regions so expression vectors provide promoter, ribosome-binding site, and terminator sequences
35
Q

WHAT ARE RECOMBINANT PROTEINS USED FOR?

A

Lab techniques–

  • ELISA–matched antibody pairs–standards
  • Western Blot–positive controls
  • Immunohistochemistry
  • Enzyme assays
  • Cellular responses to stress and disease
  • In animal models–can help with identifying therapeutic targets
36
Q

EXAMPLES OF RECOMBINANT PROTEINS

A

First use–insulin (1982)

Now–recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs,treating major diseases

Enzymes–animal feed enhancement

Lactic acid bacteria–used for fermenting foods, now adapted for use in human/animal digestion and nutrition

37
Q

ADVANTAGES OF RECOMBINANT PROTEINS

A

Ethical considerations

Quick

Cost

Scaling

38
Q

DISADVANTAGES OF RECOMBINANT PROTEINS

A

Contamination, e.g. proteasesInactive protein, e.g. inclusion bodies

Small proteins only

Lack of post-translational modifications

39
Q

What occurs at the stationary phase?

A

catch different parts of solution or gas we are measuring eg. paper where we pass liquid over it

40
Q

What occurs at the column?

A

load sample with solution

let it run through

41
Q

What is column packing?

A

the thing that is absorbing packing material

42
Q

How do you equilibrate a column?

A

we need to equilibrate column

this is done by putting solvent through column eg. water or petrol

so column is bathed in whatever the solute is going to dissolve in

43
Q

What is silica good for?

A

polar compounds

44
Q

What effects does dialysis use?

A

uses osmotic effects to draw out small solutes

45
Q

What solid phase is used in ion exchange chromatography?

A

resin - charged

46
Q

How do proteins move in ion exchange chromatography?

A

proteins move through depending on charge

47
Q

What happens if we have a negative charge on our resin (cation exchange) - ion exchange chromatography

A

proteins with negative charge move faster ad come out earlier

48
Q

How does size-exclusion chromatography separate proteins?

A

separates protein molecules by size
- larger molecules come out earlier as smaller molecules are trapped in the beads

49
Q

How does affinity chromatography bind to molecules?

A

by forming ligands
- they trap molecules of interest as it goes down the column

50
Q

What does gradient elution involve?

A

change binding conditions

proteins separated out differently

2 peaks formed

51
Q

Matrix - Affinity Chromatography

A

for ligand attachment

matrix should be chemically and physically inert

52
Q

Spacer Arm - Affinity Chromatography

A

used to improve binding between ligand and target molecules by overcoming any effects of steric hindrance

53
Q

Ligand - Affinity Chromatography

A

molecule that binds reversibly to a specific target molecule or group of target molecule

54
Q

Affinity Chromatography - Step 1

A

affinity medium is equilibrated in binding buffer

55
Q

Affinity Chromatography - Step 2

A

sample is applied under conditions that favour specific binding of the target molecules(s) to a complementary binding substrate (the ligand)

target substance bind specifically but reversibly to the ligand and unbound material washes through the column

56
Q

Affinity Chromatography - Step 3

A

target protein is recovered by changing conditions to favour elution of bound molecules

elution is performed specifically using a competitive ligand or non-specifically by changing the pH, ionic strength or polarity

target protein is collected and purified, concentrated form

57
Q

Affinity Chromatography - Step 4

A

affinity medium is re-equilbrated binding buffer

58
Q

Process of affinity chromatography

A

equilibrium

adsorption of sample and elution of unbound material

wash away unbound material

elute bound protein(s)

re-equilibration

59
Q

What happens in Hydrophobic Interaction Chromatography (HIC)

A

salt concentration is lowered gradually and samples will elute from column in order of hydrophobicity

60
Q

What is on the y axis of a chromatography analysis graph?

A

absorbance

61
Q

What is on the x axis of a chromatography analysis graph?

A

column volumes (CV)

62
Q

What is added to a Hydrophobic Interaction Chromatography (HIC) and why?

A

as we move along, more tightly bound molecules
therefore we change salts

63
Q

Why do we add Ammonium Sulphate to Hydrophobic Interaction Chromatography (HIC)?

A

helps precipitate out any addition of proteins

64
Q

What does increasing salt concentration on Hydrophobic Interaction Chromatography (HIC) allow?

A

better binding

65
Q

Tag

A

bind gene encoding on target protein so we recognise it at the end

tag can be put at either ends

66
Q

Why do we use tags?

A

we use its properties to separate our target

67
Q

Polyarylamide gel

A

acts as molecular signal to filter different proteins

68
Q

SDD

A

type of detergent

69
Q

Page

A

type of polyacrylamide gel

70
Q

What direction foes SDS-PAGE run?

A

run vertically rather than horizontally

71
Q

Kamasi Blue

A

very good for binding to protein

tells us where proteins are coming out of gel

72
Q

What can we use to look at where protein is sitting and what the isoelectric point is?

A

staining

73
Q

What is the Western blotting?

A

bind proteins of interest with primary antibodies and a conjugate with a dye

74
Q

What does the plasmid vector do in recombinate protein chromatography?

A

take sequence and amplified part of reproductive processes

75
Q

Where do you get recombonant proteins from?

A

from humans

76
Q

What do we need if we put a sequence of interest into vector?

A

we need promotor regions

77
Q

What’s a disadvantage of sequence of interest into vector?

A

cDNA does not have regulatory genes

78
Q

How do beads in the column attach in affinity chromatography?

A

Beads in the column - covalently attached ligand

79
Q

Process of affinity chromatography

A

Any protein with affinity for the ligand binds to the beads - migration retarded

Proteins that do not bind flow more rapidly through the column

Bound proteins eluted by a solution containing either a high concentration of

Free ligand competes with the ligand attached to the beads, releasing the protein product that elutes from the column is often bound to the ligand used

Analytical and Clinical (11 decks)

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