Protein analysis

Tools for protein analysis

Progress in science depends on new techniques, new discoveries and

new ideas, probably in that order.
Sydney Brenner

8/23/2017 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.

8/23/2017 by TDLV 2
 Main Steps in Protein Analysis

1. Extraction of proteins.

2. Purification of the protein.

3. Structural Characterization of the protein.

8/23/2017 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

8/23/2017 by TDLV 4
 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

8/23/2017 by TDLVis revealed by its elution volume
5
8/23/2017 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.

8/23/2017 by TDLV 7
 Chromatography for protein purification

8/23/2017 by TDLV 8
 Chromatography-3 matrices

8/23/2017 by TDLV 9
 Protein purification by chromatography

8/23/2017 by TDLV 12
 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

8/23/2017 by TDLV 13
 Purification of protein complexes by using a GST-
tagged fusion protein

8/23/2017 by TDLV 14
 Protein analysis
SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel electrophoresis
Protein separation

8/23/2017 by TDLV 15
 SDS PAGE

8/23/2017 by TDLV 16
 Separation of proteins by isoelectric point

8/23/2017 by TDLV 17
 Specific proteins can be detected by Western blotting

8/23/2017 by TDLV 18
 The diffraction of X-ray by protein crystals can reveal a
proteins exact structure

8/23/2017 by TDLV 19
Determing Primary Structure of an
Entire Protein