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 the
structure of proteins

How proteins bind to substrates and other
molecules to perform their functions.

Understand the function of the protein based on its
structure.

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 their
number.
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 acids separated 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
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Genetically-engineered tags provide an easy way to
purify proteins
- antigenic determinant, or epitope, recognized
by a specific antibody

- short string of histidines (His-tag) binds to an
affinity column containing immobilized nickel
ions

- Glutathione S-transferase (GST) fusion
protein binds to an affinity column containing
glutathione

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Purification of protein complexes by using a GST-
tagged fusion protein
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Protein analysis
SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel electrophoresis
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
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The diffraction of X-ray by protein crystals can reveal a
proteins exact structure