Protein Purification

Why do we
need purified
protein ?
Why we need purification protein?

Secondary protein structure Quaternary protein structure

Amino acid sequence linked by Protein consist with more than
hydrogen bonds one amino acid chain

Primary protein structure Tertiary protein structure

Amino acid sequence Actual functional unit of the protein
How could we
know we
isolate the
particular
protein we
need?
How to identify target protein?

Western Blot Assay

Enzyme Activity Assay

Let’s start to purify the
target protein !
Step 1. Proteins Release

On the laboratory scale:

 Sonication

 French press

 Enzymatic or chemical
cleavage of the cells

 Freeze–thawing
Step 1. Proteins Release

For larger scale:

 High pressure
homogenization

 Bead milling
Step 2. Gradient Centrifugation

The mixture is fractionated by centrifugation. We need to make

sure the target protein enriched in which component of the
fractions by assays.
 “
Proteins could be purified according to their basic characteristics,
such as solubility, size, charge, and specific binding affinity.

Usually, protein mixtures are subjected to a series of separations,

each based on a different property to yield a pure protein.
Step 3. Salting Out
Separation by
Different
Solubility
Solubility

Supernatant

Precipitate

Salt Concentration
Step 4. Dialysis

Smaller
Proteins molecules
and ions

This process could not

distinguish between proteins
effectively.
Step 5. Chromatography

1. Gel-Filtration
Chromatography
(by size)
Step 5. Chromatography

2. Ion-Exchange
Chromatography
(by charge)

cellulose
/agarose

Carboxylate(CM)
groups cellulose
/agarose
Diethylaminoethyl
(DEAE) groups
Step 5. Chromatography

2. Ion-Exchange
Chromatography
(by charge)
Elute negatively
charged protein with
salt solution
(NaCl)
Step 5. Chromatography

3. Affinity Chromatography
(by specific binding affinity)
Step 5. Chromatography
3. Affinity Chromatography
GC box
DNA
Mixture of
proteins
(by specific binding affinity)
Agarose High salt buffer
bead

transcription
factor

Other proteins Purified protein

flow through
Step 5. Chromatography
3. Affinity Chromatography
Mixture of
proteins
(by specific binding affinity)
GC box
High salt DNA
buffer

Agarose
bead

1. loading
2.
3. attaching
thethe
eluting the chemical
sample
target intogroup
proteinthe
by
which
column,
high could affinity
and let with target
the target
concentration salt
proteinaffinity
protein
solution to the column.
with chemical
transcription group.
factor

Other proteins
Purified protein
flow through
Step 5. Chromatography

4. High-Pressure Liquid
Chromatography

Column materials are

much more finely divided -
more interaction sites
Step 5. Chromatography

4. High-Pressure Liquid Chromatography

Step 6. Quantity and Quality Analysis

1. Electrophoresis
Step 6. Quantity and Quality Analysis
Ion Molecular
Salt exchange exclusion Affinity
Homogenate Fractionation chromatography chromatography chromatography

1. Electrophoresis

The initial fractions

will display dozens to
hundreds of proteins.

As the purification
progresses, the
number of bands will
diminish.
Step 6. Quantity and Quality Analysis

1. Electrophoresis

pH10
Stable pH Gradient

pH 3
Step 6. Quantity and Quality Analysis

1. Electrophoresis
Step 6. Quantity and Quality Analysis

2. Enzyme activity
assay and SDS-PAGE

Enzyme Activity Assay SDS-PAGE

 “
 A good purification scheme takes into account both
purification levels and yield.
 A high degree of purification and a poor yield leave little protein
with which to experiment.
 A high yield with low purification leaves many contaminants in
the fraction and complicates the interpretation of experiments.
Step 7. Quantity and Quality Analysis

3. Mass spectrometry

Matrix-assisted laser
desorption-ionization (MALDI)
and electrospray
spectrometry
Contact Information:

Phone: 1-631-559-9269
1-631-448-7888
Fax: 1-631-938-8127
Address: 45-1 Ramsey Road, Shirley,
NY 11967, USA
Email: info@creative-biomart.com
Thanks!
Any questions?
You can find me at contact@creative-biomart.com