Crude Extract: Proteins Can Be Separated and Purified

Exploring Proteins
• The purification of proteins is an essential first step in understanding their
function.
• The source of a protein is generally tissue or microbial cells.
• The first step in any protein purification procedure is to break open these
cells, releasing their proteins into a solution called a crude extract.
Proteins Can Be Separated and Purified

Once the extract is ready, various methods are available for purifying one
or more of the proteins it contains.
Proteins can be separated and purified according to size, charge, solubility
and binding affinity.
Protein mixtures are subjected to a series of separations, each based on

a different property to yield a pure protein.
A variety of purification techniques are available:
Salting Out
• The principle of separation is differences in protein solubility
• Most proteins are less soluble at high salt concentrations, an effect
called salting out.
• The salt concentration at which a protein precipitates differs from one
protein to another.
• Hence, salting out can be used to fractionate proteins.
Example: Fibrinogen, a blood-clotting protein, precipitates at 0.8 M

ammonium sulfate
Serum albumin precipitate at 0.2 M ammonium sulfate
Salting out is also useful for concentrating dilute solutions of proteins.

Dialysis
• The principle of separation is protein size
• Dialysis is a procedure that separates
proteins from small molecules
using a semipermeable membrane,
such as cellulose membrane with pores.
• Molecules having dimensions significantly
greater than the pore diameter are retained
inside the dialysis bag.
• Smaller molecules and ions traverse the
pores of such membrane and emerge in
the dialysate outside the bag. Dialysis:
Protein molecules (red) are retained
within the dialysis bag, whereas
small molecules (blue) diffuse into
the surrounding medium.
This technique is useful for removing a salt or other small molecules.

However, it does not distinguish between proteins effectively.
Column Chromatography
The most powerful methods for fractionating proteins.
A wide range of chromatographic procedures makes use of differences
in size, charge, binding affinities, and other properties.
Size-exclusion or Gel-Filtration chromatography (on the basis of size)
Ion-Exchange Chromatography (on the basis of charge)
Affinity Chromatography (on the basis of high affinity for specific

chemical group)
High-Pressure or High-Performance Liquid Chromatography (HPLC)

(rapid and greater resolving power of all chromatography)
Size-exclusion or Gel-Filtration chromatography
• More discriminating separations
on the basis of size
• The sample is applied to the top
of a column consisting of porous
beads made of an insoluble but
highly hydrated polymer such as
dextran or agarose (which are
carbohydrates) or polyacrylamide.
Sephadex, Sepharose, and Bio-gel
are commonly used commercial

preparations.
• Small molecules can enter these
beads, but large ones cannot.
• Large molecules flow more
rapidly through this column and
emerge first
• Small proteins enter the cavities,
Ion-Exchange chromatography
• Separation on the basis of net charge
• Protein with a net positive charge
will bind to a column of beads
containing –COO- groups; a
negatively charged protein will not
• A positively charged protein can be
eluted (released) by increasing the
concentration of sodium chloride or
another salt in the eluting buffer.
• Proteins that have a low density of
net positive charge will tend to
emerge first, followed by those
having a higher charge density.
• carboxymethyl-cellulose (CM-
cellulose) columns -cationic proteins
• diethylaminoethyl-cellulose (DEAE-
cellulose) columns -anionic proteins

Affinity chromatography
• is based on the binding affinity of a
protein.
• the beads in the column have a
covalently attached chemical group.
• a protein with affinity for this
particular chemical group will bind
to the beads in the column, and its
migration will be retarded.
• the desired protein is eluted by
adding a high concentration of a
soluble chemical group or altering
the conditions to decrease binding
affinity.
Affinity chromatography
Example:
• purifying the plant protein concanavalin A
- has affinity for glucose
- can be purified by passing a crude extract through a column of
beads containing covalently attached glucose residues.
- concanavalin A binds to such a column, whereas most other
proteins do not.
- the bound concanavalin A can then be released from the column
by adding a concentrated solution of glucose. The glucose in
solution displaces the column-attached glucose residues from
binding sites on concanavalin A.
• isolating transcription factors
- a protein mixture is percolated through a column containing
specific DNA sequences attached to a matrix;
- proteins with a high affinity for the sequence will bind and be
retained.
- the transcription factor is released by washing with a solution
containing a high concentration of salt.
HPLC, or high-performance liquid chromatography
• an enhanced version of the column techniques
• the column materials are much more finely
divided; there are more interaction sites and thus
greater resolving power.
• HPLC makes use of high-pressure pumps that
speed the movement of the protein molecules
down the column.
• the liquid mobile phase is forced through the
stationary phase using pressure.
• by reducing the transit time on the column,
HPLC can limit diffusional spreading of protein
bands and thus greatly improve resolution.
A simple HPLC include

a solvent reservoir, a pump, injector,
a column, detector, recorder

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Exploring Proteins

• The purification of proteins is an essential first step in understanding their

Proteins Can Be Separated and Purified

Protein mixtures are subjected to a series of separations, each based on

Example: Fibrinogen, a blood-clotting protein, precipitates at 0.8 M

Salting out is also useful for concentrating dilute solutions of proteins.

This technique is useful for removing a salt or other small molecules.

Size-exclusion or Gel-Filtration chromatography (on the basis of size)

Ion-Exchange Chromatography (on the basis of charge)

Affinity Chromatography (on the basis of high affinity for specific

High-Pressure or High-Performance Liquid Chromatography (HPLC)

Sephadex, Sepharose, and Bio-gel

are commonly used commercial

cellulose) columns -anionic proteins

A simple HPLC include

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