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Protein analysis
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Progress in science depends on new techniques, new discoveries
and
new ideas, probably in that order.
Sydney Brenner
Tools for protein analysis
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Why do we analyse proteins?
Explore how amino acid sequences specify thestructure of
proteins
How proteins bind to substrates and othermolecules to perform
their functions.
Understand the function of the protein based on its
structure.
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Determing Primary Structure of an
Entire Protein
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Main Steps in Protein Analysis
1. Extraction of proteins.
2. Purification of the protein.
3. Structural Characterization of the protein.
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Protein Characterization
Characterization of proteins and peptides: 3
different processes:
1. Determining the Amino Acid Composition
finding out the amino acids that make up the protein and
theirnumber.
2. Determining the Amino Acid Sequence
finding out the sequence of amino acids of the proteins in
their
order.
3. Determining the Molecular mass of the Protein
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Determination of Amino Acid Composition
peptide hydrolyzed amino acids by heating in 6M HCl at
110C for 24 hrs. The amino acidsseparated by HPLC.
measured by reacting them with ninhydrin. Alpha Amino acids
intense blue color , imino acids (proline, hydroproline)yellow
color.
The concentration of a single amino acid is proportional to
the
light absorbance of the solution after adding ninhydrin.
The elution profile of the amino acids is obtained.
The identity of the amino acid is revealed by its elution
volume
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Determination of protein Molecular Mass by Mass Spectrometry
Mass Spectrometry: include an ionization source, a mass
analyzer and a detector. The ion hits the mass analyzer. The
time the ions take in flight is recorded by triggering a
laser
pulse.
It depends on one principle
The smaller ions will fly faster and hit the detector first
then the larger ions reach. The ions hit the detector in
order by mass.
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Chromatography for protein purification
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Chromatography-3 matrices
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Protein purification by chromatography
G ti i t i t
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Genetica y-engineere tags provi e an easy way to
purify proteins
- antigenic determinant, or epitope, recognized
by a specific antibody
- short string of histidines (His-tag) binds to anaffinity
column containing immobilized nickel
ions
- Glutathione S-transferase (GST) fusion
protein binds to an affinity column containing
glutathione
P i i ti t i i GST
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Puri ication o protein comp exes y using a GST-
tagged fusion protein
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Protein analysis
SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel
electrophoresi
Protein separation
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SDS PAGE
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Separation of proteins by isoelectric point
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Specific proteins can be detected by Western blotting
T e i raction o X ray y protein crysta s can revea a
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T e i raction o X-ray y protein crysta s can revea aproteins
exact structure
