Protein purification techniques

1. Protein
Purification
Techniques
Navaira Arif
Roll No. 18

2. The basic aim in protein
purification is to isolate one
particular protein of interest from
other contaminating proteins to
study its structure and function,
increasing its stability and large
scale production

3. Protein Purification
• Purification of proteins is an essential first step
in understanding their function
• Proteins must be released from the cell to be
purified
• Based on the basic properties of proteins like
solubility, size, charge and binding affinity

4. General Steps in Protein Purification
Selection of a
protein source
Assay of
Proteins
Homogenization
and
Solubilization
Stabilization of
Proteins
Detergent e.g.
Triton X 100
pH 7 and
temperature
below 25°C
Radioimmunoassa
y, ELISA, Western
Blotting

5. 1. Ammonium Sulfate Precipitation
• This technique exploits the fact that the
solubility of most proteins is lowered at high
salt concentrations
• The concentration of ammonium sulfate at
which a particular protein comes out of
solution and precipitates is different from
other proteins in the mixture

6. 2. Dialysis
• A semipermeable membrane is used to
remove small molecules such as salts and
ammonium sulfate from a protein solution
• Based upon size of molecules

7. 3. Gel Filtration Chromatography
• separate proteins according to their size. Also
termed as “size exclusion chromatography”
• A gel filtration column has beads with
channels running through them e.g. agarose
• A good gel for gel filtration contains about
95% water

8. • Smaller molecules
can freely enter the
internal solvent
space of the gel
bead
• Larger molecules are
too large to
penetrate the gel
pores and travel
between beads and
elute first

9. 4. Ion exchange chromatography
• separate proteins on basis of their over all
(net) charge
• Retention is based on electrostatic interaction
between solute ions and fixed ion charge on
the stationary phase
Cation Exchange
Chromatography Anion Exchange
Chromatography

10. Anion exchange resins
(positive charge) separate
negatively charged
compounds
Cation exchange resins
(negative charge) separate
positively charged
compounds

13. 5. Affinity chromatography
• Based upon molecular conformation
• Exploits the specific, high affinity, non-
covalent binding of a protein to another
molecule, the ligand
• Ligands function in a fashion similar to
that of antigen-antibody interactions

14. The ligand is immobilized onto a
solid support matrix
The crude extract is passed
through the column.
The target molecule for which
the ligand possesses affinity is
retained
All other material is eluted
The bound target protein is
eluted by alteration of the
mobile-phase conditions.

15. 6. Fast Performance Liquid
Chromatography
• A type of liquid chromatography where the
flow rate of the solvent is set through
computer input and controlled by pumps
• The chromatographic bed is composed by the
gel beads alone when they are inside the
column
• The sample is introduced into the injector and
then carried into the column by the flowing
solvent

16. Once in the column,
the sample mixture
separates as a result
of different
components adhering
to or diffusing into
the gel in form of
different zones called
“Bands”

17. 7. Gel Electrophoresis
• When placed in an electric field, proteins
(having net charge) will move towards one
electrode or the other
• The greater the net charge the faster the
molecule will move
• PAGE – Polyacrylamide Gel Electrophoresis
(carried out in a gel which serves as a
molecular sieve)

18. Protein samples are loaded into sample wells
An
electric
field is
applied
across
the gel
from
top to
bottom
Proteins
migrate
down
through
the gel
The smaller the protein the further it will migrate

19. SDS- PAGE
• Protein mixture is
heated in the presence
of 2-mercaptoethanol
and SDS
• Unfolded polypeptide
chains will then migrate
towards the anode
• Smaller polypeptides
migrate further through
the gel than larger ones

20. Proteins are purified from a mixture using combination
of several techniques based on protein properties