Chapter 2 4 protein analysis

Protein analysis

Tools for protein analysis

Progress in science depends on new techniques, new discoveries and
new ideas, probably in that order.
Sydney Brenner
04/25/2023

by TDLV

1


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.

04/25/2023

by TDLV

2


Main Steps in Protein Analysis

1. Extraction of proteins.
2. Purification of the protein.
3. Structural Characterization of the protein.

04/25/2023

by TDLV

3


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

04/25/2023

by TDLV

4


Determination of Amino Acid Composition

• peptide  hydrolyzed  amino acids by heating in 6M HCl at 110ºC
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
by TDLVis revealed by its elution volume
5

04/25/2023


04/25/2023

by TDLV

6

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.

04/25/2023

by TDLV

7


Chromatography for protein purification

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8


Chromatography-3 matrices

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9


Protein purification by chromatography

04/25/2023

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10


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

04/25/2023

by TDLV

11


Purification of protein complexes by using a GST-tagged fusion protein

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12


Protein analysis

 SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel electrophoresis

Protein separation

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by TDLV

13


SDS PAGE

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14


Separation of proteins by isoelectric point

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15


Specific proteins can be detected by Western blotting

04/25/2023

by TDLV

16

The diffraction of X-ray by protein crystals can reveal a protein’s exact structure

04/25/2023

by TDLV

17


Determing Primary Structure of an Entire
Protein