Biochemistry

Dr. Tareq Alhindi

Lecture Notes (6-2)

 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
Chromatography
The mixture is dissolved in a fluid called the mobile phase, which carries it
through a structure holding another material called the stationary phase.
The various constituents of the mixture travel at different speeds, causing
them to separate. The separation is based on differential partitioning
between the mobile and stationary phases.

Chromatography methods allow us to sort proteins by size or by how they

bind to or separate from other substances.

In column chromatography, long glass tubes are filled with microscopic

resin beads and a buffered solution. The protein extract is then added and
flows through the resin beads in a glass column.

Depending on the resin used, the protein either sticks to the beads or
passes through the column while the beads act as a filtration system.
Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
Size-exclusion chromatography (SEC) also called gel-filtration
chromatography uses gel beads as a filtering system. Larger protein
molecules quickly work their way around the gel beads while
smaller molecules pass through more slowly because they are able
to slip through tiny holes in the beads.
The gels are available in a variety of pore sizes, and the necessary
gel for proper separation depends on the molecular weight of the
contaminants or proteins being separated.
It can be used to estimate molecular weight by comparing the
sample with a set of standards. Each gel also has an exclusion limit,
a size of protein that is too large to fit inside the pores.
The chromatography column is packed with fine, porous beads
which are composed of dextran polymers (Sephadex), agarose
(Sepharose), or polyacrylamide (Sephacryl or BioGel P).
 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
Ion-exchange (IonX) chromatography, relies on an electrostatic
charge to bind proteins to resin beads in a column.
While the charged proteins cling to the resin, other contaminants
pass through and out of the column.

A negatively charged resin is a cation exchanger, and a positively

charged one is an anion exchanger.
 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
The proteins can then be eluted (released from the resin) by changing the electrostatic charge; this is done by rinsing the
column with salt solutions of increasing concentrations. The bound protein is then released from its attachment (detected by
viewing under UV 280) and collected.
 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells
Affinity chromatography relies on the ability of most proteins to bind
specifically and reversibly to uniquely shaped compounds called
ligands.
Ligands are small molecules that bind to a particular large molecule
in a protein.
After the proteins have bound to the resin beads, a buffer solution is
used to wash out the unbound molecules.

Special buffer solutions are used to cause desorption (to break the
ligand bonds) of the retained proteins (change in pH or ionic
strength).
The bound protein can be eluted from the column by adding high
concentrations of the ligand in soluble form, thus competing for the
binding of the protein with the stationary phase.
 Protein Purification and
Characterization Techniques
Extracting Pure Proteins from Cells
Immobilized metal affinity chromatography (IMAC) is a specialized variant of affinity
chromatography where the proteins or peptides are separated according to their
affinity for metal ions that have been immobilized by chelation to an insoluble matrix.

In hydrophobic interaction chromatography (HIC), proteins are sorted on the basis of

their repulsion of water. The column beads in HIC are coated with hydrophobic
molecules, and the hydrophobic amino acids in a protein are attracted to the similar
chemicals in the beads.
 Protein Purification and Characterization Techniques
Extracting Pure Proteins from Cells

High-performance liquid chromatography (HPLC)

Previously described chromatography depend on

gravity flow or very low pressure pumps to move the
extract through the columns. Such low-flow methods
can take several hours to process a single sample.

In contrast, HPLC systems use greater pressure to

force the extract through the column in a shorter
time.
HPLC systems have limitations, less protein is
separated, so the technique is more useful in
analytical situations than in mass production.

Reverse phase HPLC is a widely used technique for the

separation of nonpolar molecules.