Panel 4–6 Protein separation by electrophoresis

167

protein with two

GEL ELECTROPHORESIS The detergent CH3 subunits, A and B,
sodium dodecyl
sulfate (SDS) joined by a disulfide single subunit
sample loaded onto gel CH2 bridge
is used to protein
by pipette
cathode
solubilize CH2 A B C
proteins for SDS
plastic casing
polyacrylamide- CH2 S-S
gel electrophoresis.
CH2
CH2
CH2
HEATED WITH SDS AND MERCAPTOETHANOL
CH2
_
__ __ __ _ __
buffer CH2 __ ___ ___ ___ ___ __
___ _ _ _ __ _____ ___ __ _ _ _
_ _ _ __ _ _ __
CH2 _ _ __ __ _SH__ ____ __ __ _ _ ___ _ _ _ _ _ ___
gel + anode ___ __ __ _____ __ __
__ _ _ __
_ __ _
__ _ __ __ __ __
CH2 __ _ ___ _HS _ _
__ _ __ _ _ _ ___
_
_____ __ _ _ ___ ___ ___ negatively __ _ _ _
__ _ _ _ _ ___ _
CH2 _ _ ___ __ _
_ _ __ _ charged SDS C
_ _ __ molecules
O A B

buffer
When an electric field is applied to a solution
containing protein molecules, the molecules
will migrate in a direction and at a speed that
reflects their size and net charge. This forms
the basis of the technique called
electrophoresis.
ISOELECTRIC FOCUSING
For any protein there is a characteristic
pH, called the isoelectric point, at which
the protein has no net charge and
therefore will not move in an electric
field. In isoelectric focusing, proteins
are electrophoresed in a narrow tube of
polyacrylamide gel in which a pH
gradient is established by a mixture of
special buffers. Each protein moves to a
point in the gradient that corresponds
to its isoelectric point and stays there.
stable pH gradient
10 9 8 7 6 5 4
+
+++
+++
O S O POLYACRYLAMIDE-GEL ELECTROPHORESIS
O Na +
SDS
SDS polyacrylamide-gel electrophoresis B
(SDS-PAGE) Individual polypeptide chains form a
complex with negatively charged molecules of C
sodium dodecyl sulfate (SDS) and therefore migrate
as a negatively charged SDS–protein complex
through a slab of porous polyacrylamide gel. The
A
apparatus used for this electrophoresis technique
is shown above (left ). A reducing agent
(mercaptoethanol) is usually added to break any
–S–S– linkages in or between proteins. Under +
these conditions, proteins migrate at a rate that slab of polyacrylamide gel
reflects their molecular weight.
TWO-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS
Complex mixtures of proteins cannot be resolved well on one-dimensional gels, but
two-dimensional gel electrophoresis, combining two different separation methods, can
be used to resolve more than 1000 proteins in a two-dimensional protein map. In the
first step, native proteins are separated in a narrow gel on the basis of their intrinsic
charge using isoelectric focusing (see left ). In the second step, this gel is placed on top of
a gel slab, and the proteins are subjected to SDS-PAGE (see above ) in a direction

perpendicular to that used in the first step. Each protein migrates to form a discrete spot.
+

___
+

_ _
___ All the proteins in stable pH gradient acidic
basic
an E. coli bacterial
At high pH, At low pH,
the protein
cell are separated in
the protein
100
SDS migration (mol. wt. x 10–3)

is negatively is positively this 2-D gel, in

charged. charged. which each spot
corresponds to a
++ _
+_ _+ different
__ + polypeptide chain. 50
+

_+ _ They are separated

__+
according to their
isoelectric point
from left to right 25
_+_
+_ _+ and to their
+ molecular weight
+

_+_ from top to

+_ _+
+ bottom. (Courtesy
of Patrick O'Farrell.)
The protein shown here has an isoelectric pH of 6.5.