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Techniques of Protein Purification

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Chapter 6 Techniques of Protein Purification You should become familiar with this entire chapter so that you can use it for reference. But you are only responsible ... – PowerPoint PPT presentation

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Title: Techniques of Protein Purification


1
Chapter 6
Techniques of Protein Purification
You should become familiar with this entire
chapter so that you can use it for reference.
But you are only responsible for the topics in
the chapter that were covered in lecture.
2
  • Protein isolation
  • Selection of protein source
  • Tissues from animals
  • Microorganisms (E. coli or yeast)
  • Molecular cloning techniques
  • Methods of solubilization
  • Osmosis lysis (with hypotonic solution)
  • Use of lysozyme (enzyme that degrades cell wall)
  • French press or sonication

3
Stabilization of proteins 1. pH (think
buffers!) 2. Temperature (close to 0oC) Thermal
stability could be used for purification 3.
Addition of protease inhibitors 4. Gentle
handling (no frothing) Assay of proteins 1. If
purifying an enzyme, use the reaction it
catalyzes as an assay 2. If metalloprotein use
the metals to follow the protein 3.
Immunochemical techniques (antibodies)
4
General strategy of protein purification
Proteins are purified by fractionation
procedures, a series of independent steps in
which the properties of protein of interest are
utilized to separate it from other contaminating
proteins. How do we know our sample of protein
is pure? We don't! The best we can do is to
demonstrate by all available methods that our
sample consists of only one component.
5
General strategy of protein purification
Characteristic Procedure Solubility 1.
Salting in 2. Salting out Ionic charge 1.
Ion exchange chromatography 2.
Electrophoresis 3. Isoelectric
focusing Polarity 1. Adsorption
chromatography 2. Paper chromatography 3.
Hydrophobic interaction chromatography Molecular
size 1. Dialysis and ultrafiltration 2.
Gel electrophoresis 3. Gel filtration
chromatography 4. Ultracentrifugation Binding
specificity 1. Affinity chromatography
6
  • Solubility of a protein in aqueous solution
  • Depends strongly on
  • Concentrations of dissolved salts
  • pH
  • Temperature
  • 4. Addition of water-miscible organic solvents,
    e.g., ethanol or acetone

7
Solubility of carboxyhemoglobin at its
isoelectric point as a function of ionic strength
and ion type
Page 131
8
Solubilities of several proteins in ammonium
sulfate solutions
Page 131
9
Solubility of b-lactoglobin as a function of pH
at several NaCl concentrations
Page 132
10
Isoelectric Points of Several Common
Proteins Protein pI Pepsin
1.0 Ovalbumin (hen) 4.6 Serum albumin
(human) 4.9 Tropomyosin 5.1 Insulin
(bovine) 5.4 Fibrinogen (human)
5.8 g-Globulin (human) 6.6 Collagen
6.6 Myoglobin (horse) 7.0 Hemoglobin
(human) 7.1 Ribonuclease A (bovine)
9.4 Cytochrome c (horse) 10.6 Histone
(bovine) 10.8 Lysozyme (hen) 11.0 Salmine
(salmon) 12.1
11
Protein crystals
Page 133
12
Chromatographic separations
Protein separation and purification by column
chromatography
From Lehninger Principles of Biochemistry
13
Column Chromatography Ion Exchange
From Lehninger Principles of Biochemistry
14
(No Transcript)
15
A schematic diagram illustrating the separation
of several proteins by ion exchange
chromatography using stepwise elution
16
Column Chromatograph Size-exclusion
Gel filtration chromatography can be used to
estimate molecular masses
From Lehninger Principles of Biochemistry
17
A schematic illustration of gel filtration
chromatography
18
The separation of macromolecules by affinity
chromatography
19
Hydrophobic interaction chromatography Proteins
contain hydrophobic amino acid side-chains, some
of which are exposed at the surface of the
protein. Proteins will therefore often bind to
other hydrophobic molecules. Hydrophobic
interaction columns are produced by covalently
attaching hydrophobic molecules such as acyl
chains or phenyl groups to insoluble carbohydrate
resins.
20
Dialysis is a form of molecular filtration
21
Protein separation and characterization by
Electrophoresis
Migration of ions in an electric field is widely
used for the analytical separation of
biomolecules.
22
Polymerization of acrylamide and
N,N-methylenebisacrylamide to form a
cross-linked polyacrylamide gel
Page 146
23
  • SDS binds to proteins in amounts roughly
    proportional to molecular weight.
  • The high negative charge of the SDS-protein
    complex is greatly in excess of the inherent
    charge of the protein.
  • Electrophoresis in the presence of SDS therefore
    separates almost exclusively by molecular weight.

24
  • Polyacrylamide gel
  • Stain proteins to visualize, e.g., Coomassie
    blue
  • (see page 93)
  • Rate of migration depends roughly on
    charge-to-mass ratio.
  • (Protein shape may also influence migration
    rate.)

Larger proteins move more slowly Smaller
proteins move faster
From Lehninger Principles of Biochemistry
25
From Lehninger Principles of Biochemistry
26
A logarithmic plot of the molecular masses of 37
different polypeptide chains ranging from 11 to
77 kD versus their relative electrophoretic
mobilities on an SDS-polyacrylamide gel
27
Detection of proteins by immunoblotting (aka
Western Blot)
Page 148
28
Setting up a purification Table
Total Enzyme Activity the activity per ml of
enzyme solution multiplied by the total volume of
that fraction. Total Protein the protein
concentration per ml of solution multiplied by
the total volume of that fraction. Specific
activity Total Activity/Total Protein or
Activity/Protein concentration Fold-purification
(Specific activity at a given step)/(Specific
activity of starting sample) Yield (Total
activity at a given step) / (Total activity of
starting sample)100
29
Setting Up a Purification Table
Total Enzyme Activity the activity per ml of
enzyme solution multiplied by the total volume of
that fraction. (If you have 50 ml of the original
homogenate, the volume is 50 ml) Total Protein
the protein concentration per ml of solution
multiplied by the total volume of that
fraction. Specific activity Total
Activity/Total Protein or Activity/Protein
concentration Fold-purification (Specific
activity at a given step)/(Specific activity of
starting sample) Yield (Total activity at a
given step)/(Total activity of starting
sample)100
30
From Lehninger Principles of Biochemistry
31
Purification of Rat Liver Glucokinase
Page 142
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