DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 16

be used as probe alternatives to antibodies for western blotting. Com-
mon lectins used for these studies include concanavalin A, a lectin iso-
lated from jack beans which binds mannose residues, and ricin, a lectin
from castor beans which binds galactose residues. Ricin is extremely
toxic, so much so that it is considered a prime bioterrorist weapon.
High-performance liquid chromatography (HPLC)
HPLC is a generic term that refers to chromatography using fully
automated equipment and “high performance” adsorbents for isolating
and purifying compounds. The most common form of HPLC is reversed
phase HPLC (RP-HPLC), which has very high resolving power for pep-
tides and proteins up to 100 kiloDaltons (kDa), although it is best used
for proteins of 30 kDa and smaller. It is based on the use of hydrophobic
“reversed-phase” surfaces (usually silica with hydrophobic chains) that
interact with the polypeptides or proteins added to the column. When
the organic component of the mobile phase reaches a critical concen-
tration, defined by the properties of the polypeptide, it is eluted in a very
sharp peak. RP-HPLC can be used to distinguish between variants of
hormones such as insulin that differ by a single amino acid. Given its
high resolving power, it is the method of choice to purify proteins before
subjecting them to Mass Spectroscopy.
RP-HPLC can, however, cause some transient denaturation during
the adsorption and elution procedure, and therefore care must be taken
if the investigator desires fully functional proteins. It is therefore mostly
used to separate small molecules and polypeptides.
Fast protein liquid chromatography (FPLC)
FPLC is a high performance system that was designed for protein
fractionation by the company Amersham Pharmacia Biotech. FPLC uses
all of the standard protein separation methods including gel filtration,
ion exchange chromatography, affinity chromatography, etc. FPLC is
designed to improve the speed and resolution when separating proteins.
Dialysis and ultrafiltration
Once proteins have been isolated by column chromatography, they
frequently need to be stored in a new buffer or concentrated for further
use. To change the buffer, the mixture containing the protein is placed
in dialysis tubing, which is a semipermeable tubing that allows small
molecules and water to be exchanged between the contents within the
tube and the outside of the tube. By “dialyzing” the material against