Download Protein purification

PROTEIN PURIFICATION AND
IMMUNOLOGICAL METHODS
Why to purify proteins
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Activity
Post-translational modifications
Interactions and assembly
3-dimensional structure
• Analysis of biological function
• Drug development
Where to start from
• Cells and tissues
– Homogenisation, solubilisation
– Fractionation by centrifugation
– chromatography
• Cloned cDNA
– Expression of recombinant protein in bacteria, insect cells, mammalian cells or
plants
– Purification as above
– ‘Tags’ in the recombinant proteins to ease purification
You’ll also need
• Methods to assess
– Purity
– function
– yield
Properties of proteins are utilised in purification
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Proteins are polypeptides, macromolecular polymers of amino acids
Vary in net charge
Vary in size
Vary in hydrophilic or hydrophobic nature
• Separation techniques are based on these properties
Protein electrophoresis
• Proteins move through a support in electric field.
• Supporting medium is usually a gel
• Movement depends on
– Net charge of the protein
– Strength of the electric field
– Friction encountered by the protein
Polyacrylamide gel electrophoresis, PAGE
• Usually used to separate proteins
• Formed by polymerising acrylamide
• Pore size controlled by altering concentration of acrylamide => gel acts
as as support and ‘molecular sieve’
PAGE
• Native PAGE: proteins are not denatured before electrophoresis. During
electrophoresis, proteins are separated according to their net charge and
conformation (globular proteins move faster than linear ones.
• Separated native proteins may retain some of their biological activity
SDS-PAGE
• Proteins are denatured before electrophoresis by adding sodium dodecyl
sulphate and heating
• Polypeptide subunits are separated
• SDS binds to proteins giving them a large net negative charge. Original
net charge becomes irrelevant.
• Proteins are separated by size only
SDS-PAGE can be used to approximate molecular
weight
SDS-PAGE: detection of proteins
• After electrophoresis the gel can be stained with a dye that bind proteins
– Coomassie Blue
– Silver staining
• Autoradiography
• Electroblotting to membrane and detection using antibodies
Western Blotting
- a commercial break
IEF and 2-D electrophoresis
• Isoelectric focusing separates proteins according to their charge.
• PAGE gel is saturated with ampholytes, a mixture of polyanionic and
polycationic molecules. In electric field ampholytes separate and form a
gradient based on their net charge
• Ampholyte gradient establishes a pH gradient
• Proteins migrate through the gradient until they reach their pI, the pH at
which the net charge is zero
IEF and 2-D electrophoresis
IEF and 2-D electrophoresis
Proteomics
• Identification and analysis of proteins expressed in cells, tissues or
organisms
• 2-d electrophoresis,
• Mass spectrometry
• Protein microsequencing.
• Proteome is much more complex than genome
Proteomics
Time-of-Flight Mass Spectrometry
Liquid chromatography
• Molecules dissolved in solution will interact with with solid surface.
• When solution is flowing across the surface, molecules that interact
tightly or more frequently with the solid surface move more slowly than
molecules that do not interact with the solid support.
• Liquid chromatography is performed in a column packed with beads.
Liquid chromatography
• The nature of the beads in the column determines how proteins are
separated
• Gel filtration - mass
• Ion-exchange chromatography - charge
• Affinity chromatography - binding affinity
• Reverse phase - hydrophilic/hydrophobic
Cromatography column and fraction collector
• Peristaltic pump regulates the flow of buffer
• Fractions are collected for next step
• Column can be changed according to need
HPLC and FPLC
• HPLC = High Performance Liquid Chromatography (or High Pressure
LC). Tightly packed even-sized beads result in high resolution. Buffers
run using high pressure.
• FPLC = Fast Protein Liquid Chromatography. Not as high pressure as
HPLC. Good for preparative protein purification.
Purified proteins can be crystallised.
X-ray Diffraction
Antibodies against proteins can be used as tool in
research and in clinic
Common steps in immunoassays
• Block non-specific binding of antibody
• Ab-Ag binding
• Separate unbound Ab (phase separation), often done by washing
unbound antibody away
• Detection (and quantitation)
Secondary antibodies are used to increase sensitivity
ELISA
• Combines Ab specificity with sensitivity of simple spectrophotometric
enzyme assays
• Competitive ELISA
– Enzyme labelled antigen competes with antigen in patient sample (not labelled)
for limited number of Ab binding sites
• Non-competitive ELISA
– ‘Sandwich’ ELISA. Excess of Ab on solid surface binds antigen from patient
sample. Second, enzyme-labelled, Ab (against different epitope) is added.
Enzyme activity directly proportional to antigen concentration
– Note size constraints.
Example of a Western Blot
Immunofluorescence detection of protein
expression in tissues