Essential Cell Biology 5th edition

PANEL 4–5 PROTEIN SEPARATION BY ELECTROPHORESIS
167
GEL ELECTROPHORESIS
cathode
sample loaded onto gel
by pipette
plastic casing
The detergent
sodium dodecyl
sulfate (SDS)
is used to
solubilize
proteins for SDS
polyacrylamidegel
electrophoresis.
CH 3
CH 2
CH 2
CH 2
CH 2
protein with two
subunits, A and B,
joined by a disulfide
(S–S) bond
A B C
S S
single-subunit
protein
CH 2
buffer
gel
buffer
When an electric field is applied to a solution
containing protein molecules, the proteins
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 pH gradient that corresponds
to its isoelectric point and stays there.
stable pH gradient
10 9 8 7 6 5 4
_ _ _
_ _ _ _ _
At high pH,
the protein
is negatively
charged.
_
_
+ _
_ _ _ +
_ + _
+ _ _
+
+
_ + _
+ _ _
+
+
+ + +
+ +
+ + +
At low pH,
the protein
is positively
charged.
+ + _
+ _ _
+
+
+ + +
The protein shown here has an isoelectric pH of 6.5.
+ anode
O
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
O
S
O
SDS
O
+
Na
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
Individual polypeptide chains form a complex with
negatively charged molecules of sodium dodecyl
sulfate (SDS) and therefore migrate as negatively
charged SDS–protein complexes through a slab of
porous polyacrylamide gel. The apparatus used for
this electrophoresis technique is shown above (left ).
A reducing agent (mercaptoethanol) is usually
added to break any S – S linkages within or between
proteins. Under these conditions, unfolded
polypeptide chains migrate at a rate that reflects
their molecular weight, with the smallest proteins
migrating most quickly.
HEATED WITH SDS AND MERCAPTOETHANOL
_ __ ___
_ _ ___ _ __ _ ___________________
_ ______________
_ __
____ _ _ _
___ SH
_
_ __
__ __ _
HS
_ ____ _ _ _____
_ ___ _ _
__ _
_____ ___
__
________________ _ _ _ __ _ _
A
B
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
an E. coli bacterial
cell are separated
in this twodimensional
gel, in
which each spot
corresponds to a
different
polypeptide chain.
They are separated
according to their
isoelectric point
from left to right
and to their
molecular weight
from top to
bottom. (Courtesy
of Patrick O'Farrell.)
SDS migration (mol. wt. x 10 –3 )
100
50
25
basic
B
C
A
_ _ _ ____ _ _ _ _
_ _ _
_ ____ _ ____
_ _ _ __ _ ____
_ _
_
negatively
___ _ ________
charged SDS C
molecules
POLYACRYLAMIDE-GEL ELECTROPHORESIS
slab of polyacrylamide gel
stable pH gradient
+
acidic