Protein Purification Techniques

Salting Out
• Often the first step in a purification protocol and
takes advantage of the different solubilities of
proteins in differing concentrations of salt.

• The salt often used is ammonium sulphate (NH 4)2SO4

• E.g. 0.8M AS precipitates fibrinogen, whereas a

solution of 2.4M AS is required to precipitate serum
albumin
 Salting Out
• This procedure can fractionate a complex mixture
of proteins and results in a 2 to 3 fold purification

• Following salting out the pellet is collected by

centrifugation and re-suspended in buffered
medium

• Following re-suspension the salt needs to be

removed
 Removal of Salt
• Salt can be removed by
• Gel filtration
• Dialysis
 Chromatography
Chromatography is the differential separation of
sample components between a mobile and a
stationary phase
 Before undertaking chromatography a number of important
considerations need to be made

• The capacity of the matrix i.e. the measure of

the amount of protein that can be absorbed
from the solution onto a unit volume/weight of
stationary phase

• Selectivity i.e. a measure of the ability of the

purification technique to absorb a protein from
solution with a high degree of rejections of
contaminants
 Matrix Materials
• High mechanical stability – allows maximum flow rate
and minimizes pressure drop

• Good chemical stability – allows maintenance of bed

structure, reduces contamination and irreversible
binding and if necessary allows for sterilization

• Has a high capacity

• Is inert
 Flow Dynamics of the Column
• A) Long Thin Columns

• These have a low flow rate (high back pressure) and

therefore have long separation times.

• The advantages of using such systems are maximum

peak resolution, however they do have the disadvantage
of a longer run time.

• Can be problematic if the protein of interest is unstable

 Flow Dynamics of the Column
• B) Short wide columns

• These minimize gel compression and have a high

flow rate (low back pressure)

• The main advantage is the high flow rate and as

such the purification step can be quickly performed

• The main disadvantage is low resolution

Size Exclusion (Gel Filtration)
 Size Exclusion (Gel Filtration)
The volume accessible for a given species (Kav) is given by:
 
Kav = (Ve – Vo)
(Vt - Vo)
Ve = volume of elution

Vo = void volume

Vt = total volume = Vi

NB: Vol (sample) ~ 1-5% of total bed vol.

→ mol wt determination (plot Ve vs log Mol Wt. of standards)

Ion Exchange Chromatography
 Immunoblot analysis of anion-exchange fractions
obtained from His –tagged NdhJ preparation

Elution profile of the solubilised thylakoid

extract from a Q-sepharose
anion exchange column

Immunoblot analysis of pooled fractions

Using antibodies specific for NdhI and NdhJ
Affinity Chromatography

Affinity chromatography of concanavalin A

(shown in yellow) on a solid support containing
covalently attached glucose residues (G).
 Examples of Group-Specific Affinity Ligands

Ligand Target Protein

5’AMP, ATPDehydrogenases

NAD, NADP “

Protein A Antibodies

Histones DNA

Lectins Glycoproteins

Calmodulin Kinases

Cibacron BlueKinases, albumin, phosphatases,DHs

 High Pressure Liquid Chromatography (HPLC)

• HPLC is an enhanced version of column techniques

• The column materials are much more finely divided and as a consequence there
are more interaction sites and therefore greater resolving power

• Due to the finer material, pressure must be applied to the column to obtain
adequate flow rates

• The net result is rapid separation and high resolution

• This technique is not usually used to purify proteins as the high pressure can lead
to a loss of activity

• Often used as an analytical tool

 Electrophoresis
When a charged particle is placed in an electric field
it will move towards its opposite charged
electrode
 Electrophoresis
• Electrophoretic separations are nearly always
carried out in gels (or solid supports) because the
gel acts as a molecular sieve that enhances the
separation

• Molecules that are small compared with the pores

in the gel readily move through the gel, whereas
molecules much larger than the pores are almost
immobile. Intermediate size molecules move
through the gel with various degrees of facility.
Electrophoresis

Polyacrylamide gels are choice

materials to act as the supporting
media in many separations as they are
chemically inert and are readily
formed by the polymerization of
acrylamide and a small amount of
cross linker.
SDS-PAGE
 Staining
(a) Coomassie blue and (b) Solver
staining of a 12% poly acrylamide
Tris-tricine gel of the purified His-
tagged NdhJ complexes
Isoelectric Focusing
2D-Electrophoresis
Proteins from E. coli cells are
separated by two-dimensional gel
electrophoresis.

In the first dimension, (x-axis) the

proteins are separated by a pH
gradient in which each protein
migrates to its isolectric point.

The second dimension separates

proteins by size on a SDS-
polyacrylamide gel.

Each spot corresponds to a single

polypeptide.
 2D-Electrophoresis

A 460 kDa
B 330 kDa
C 150 kDa
D 100 kDa