Bectlon B-Amino aci and proteins B7

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B7 CHROMATOGRAPHY OF
PROTEINS PO

Key Notes

Gerfltretion Gel filtration chromatography separates proteins on the basis of their size and
chromatography shape using porous beads packed in a column. Large or elongated proteins
cannot enter the pores in the beads and elute from the bottom [Link] column
first, whereas smaller proteins can enter the beads, have a larger volume of
liquid accessible to them and move through the column more slowly, eluting
later. Gel filtration chromatography can be used to de-salt a protein mixture
and to estimate the molecular mass of a protein.

lonexchange In ion exchange chromatography, proteins are separated on the basis of their
chomatography net charge. In anion exchange chromatography a column containing positively
charged beads is used to which proteins with a net negative charge will bind,
whereas in cation exchange chromatography negatively charged beads are
used to which proteins with a net positive charge will bind. The bound
proteins are then eluted by adding a solution of sodium chloride or by altering
the pH of the buffer.

Affinity Affinity chromatography exploits the specific binding of a protein for another
chromatography molecule, its ligand (eg. an enzyme for its inhibitor). The ligand is
immobilized on an insoluble support and packed in a column. On adding a
mixture of proteins, only the protein of interest bind_ to the ligand. All other
p
proteins pass straight through. The bound protein is then eluted from the
immobilized ligand in a highly purified form.

Related topics Protein purification (B6) Antibodies as tools (D5)

Gel filtration In gel filtration chromatography (size exclusion chromatography or molecular

chromatography sieve chromatography), molecules are separated on the basis of their size and
shape. The protein sample in a small volume is applied to the top of a columni
of porous beads (diameter 0.1 mm) that are made of an insoluble but highly
hydrated polymer such as polyacrylamide (Bio-Gel) or the carbohydrates
dextran (Sephadex) or agarose (Sepharose) (Fig. la). Small molecules can enter
the pores in the beads whereas larger or more elongated molecules cannot. The
o
smaller molecules therefore have a larger volume of liquid accessible to them:
both the liquid surrounding the porous beads and that inside the beads. In
contrast, the larger molecules have only the liquid surrounding the beads acces-
sible to them, and thus move through the column faster, emerging out of the
bottom (eluting) first (Fig. 1a and b). The smaller molecules move more slowly
through the column and elute later. Beads of differing pore sizes are available,
allowing proteins of different sizes to be effectively separated. Gel filtration
chromatography is often used to de-salt a protein sample (for example to|
remove the ammonium sulfate after ammonium sulfate precipitation; Topic B6),
 .

Buffer added to top

Mixture of
proteins

Small
PorousK Large
molecules
beads molecules

Glass
plastic
column

is of their size and

Tube to
elongated proteins collect
tom [Link] column protein
larger volume of
ore slowly, eluting
a protein mixture
Large Small
molecules molecules
o (c)
the basis of their Ve Vez
taining positively Vo Void volume
charge will bind,
V. Elution volume
harged beads are of protein
bind. The bound
ride or by altering

otein for another Volume of eluent- Log10 molecular mass

. The ligand is
mn. On adding a Fig. 1. Gel filtration chromatography. (a) Sochematic illustration of gel filtration
indicating the separation; (c) a plot of relative elution volume versus the logarithmchromatography; (b) elution diagram
of molecular mass for known
[Link] other
proteins, indicating how the molecular mass of an unknown can be read off when its relative elution volume is knowri.
eluted from the

since the salt enters the porous beads and is eluted

late, whereas the protein
D5) does not enter the beads and is eluted
early. Gel filtration chromatography
can
also be used to estimate the molecular mass of a
protein. There is a linear rela-
tionship between the relative elution volume of a protein (V./V. where V, is
raphy or molecular the elution volume of a given
protein and V, is the void volume of the column,
is of their size and that is the volume of the solvent
space surrounding the beads; Fig. 1b) and
he of the logarithm of its molecular mass. Thus a 'standard'
top a
column? curve of V./V,
against
1soluble but highlyj log10 molecular mass can be estimated for the column using proteins of known
the carbohydrates mass. The elution volume of
any sample protein then allows its molecular mass
nolecules can enter to be estimated by reference to its
position on the standard curve (Fig. 1c).
tecules cannot. The;
lon exchange In ion
accessible to them:i exchange chromatography, proteins separated
are on the basis of
their
side the beads. In} chromatography overall (net) charge. If a protein has a net
negative charge at pH 7, it will bind to
a column containing positively
ng the beads acces-/ charged beads, whereas a protein with no charge
or a net positive
nerging out of the charge will not bind (Fig. 2a). The negatively
move more slowly bound to such a column can then be eluted charged proteins
by washing the column with an
S1zes are available,i increasing gradient (increasing concentration) of a solution of sodium chloride
ated. Gel filtrationi (Na CI ions) at the appropriate pH. The Cr ions compete with the
e (for the positively charged protein for
groups on the column. Proteins having a low density
example to
pitation; Topic B6),| of negative charge elute first, followed
by those with a higher density of nega
tive charge (Fig. 2b). Columns
containing positively charged diethylaminoethyl
 a o u d l u po
rotel

(a) Mixture of
Buffer NaCl
protelns

Positively Negatively
charged *Dcharged
proteins
beads

(OO
Glass
column
Positively
charged Negatively
proteins Tubes to- charged
collect protelns
protein

Low density
of negative Higher density
charge of negative
b Positively charge
NaCl gradient
charged
proteins
f

Volume of eluent
Fig: 2. lon exchange chromatography (a) Schematic ilustration of ion exchange
chromatography: (b) elution diagram indicating the separation of a protein of net positive
charge that does not bind to the positively charged beads and passes straight through the
column, and of two proteins with different net negative charges that bind to the positively
charged beads and are eluted on increasing the concentration of NaCl applied to the
column. The protein with the lower density of negative charge elutes earlier than the protein
with the higher density of negative charge.

(DEAE) groups (such as DEAE-cellulose or DEAE-Sephadex) are used for sepa-

ration of negatively charged proteins (anionic proteins). This is called anion
exchange chromatography. Columns containing negatively charged carboxy-
methyl (CM) groups (such as CM-cellulose or CM-Sephadex) are used for the
separation of positively charged proteins (cationic proteins). This is called cation
exchange chromatography. As an alternative to elution with a gradient of NaCl,
proteins can be eluted from anion exchange columns by decreasing the pH of the
buffer, and from cation exchange columns by increasing the pH of the buffer, thus
altering the ionization state of the amino acid side-chains (see Topic B2) and hence
the net charge on the protein.
Affinity Affinity chromatography exploits the specific, high affinity, noncovalent binding
chromatography of aprotein to another molecule; the ligand. First, the ligand is covalently attached
to an and
inert poroys mattix (such as Sepharose). The protein mixture is then
B7-Chromatography of proteins
.

(a) A Mixture of
diferent
A proteins Specific Soluble ligand
protein added
binds to
ligand

Ligand
immobilized Unwanted
on insoluble proteins pass Specific
support and straight through
packed in A protein elutes
off bound to
glass column
soluble ligand

(b)
Nonspecific,
unbound proteins

Soluble ligand
added
Specific
protein

Volume of eluent

Fig. 3. Affinity chromatography (a) Schematic diagram of affinity chromatography; (b) lution diagram
indicating that nonspecific proteins that do not bind to the immobilized ligand pass straight through the
column, while the specfic protein binds to the immobilized ligand and is eluted from the column only cn
addition of soluble ligand

passed downa column containing the immobilized ligand. The protein of interest
will bind to the ligand, whereas all other proteins pass straight through (Fig. 3).
After extensive washing of the column with buffer to remove nonspecifically
bound proteins, the bound protein is released from the immobilized ligand either
by adding soluble ligand which competes with the immobilized ligand for the
protein, or by altering the properties of the buffer (changing the pH or salt
concentration). If soluble ligand is used to elute the protein from the column,
extensive dialysis often then has to be used to remove the small ligand from the
larger protein (see Topic B6). Because this technique exploits the specific, often
unique, binding properties of the protein, it is often possible to separate the
protein from a mixture of hundreds of other proteins in a single chromatographic
step. Commonly employed combinations of immobilized ligand and protein to be
purified used in affinity chromatographic systems include an inhibitor to purify
an enzyme (see Topic C4), an antibody to purify its antigen (see Topic D5), a
hormone (e.g. insulin) to purify its receptor (see Topic E5), and a lectin (eg
concanavalin A) to purify a glycoprotein (see Topics E2 and H5).
section B - Amino acids and proteins

B8

B8 ELECTROPHORESIS OF
PROTEINS

Key Notes
Native PAGE In native polyacrylamide gel electrophoresis (PAGE) proteins are applied to a
porous polyacrylamide gel and separated in an electric field on the basis of
their net negative charge and their size. Small/more negatively charged
proteins migrate further through the gel than larger/1less negatively charged
proteins.

55PKCE In SDS-PAGE, the protein sample is treated with a reducing agent to break
disulfide bonds and then with the anionic detergent sodium dodecyl sulfate
(SDS) which denatures the proteins and covers them with an overall negative
charge. The sample is then fractionated by electrophoresis through a
polyacrylamide gel. As all the proteins now have an identical charge to mass
ratio, they are separated on the basis of their mass. The smallest proteins move
farthest. SDS-PAGE can be used to determine the degree of purity of a protein
sample, the molecular mass of a protein and the number of polypeptide.
subunits in a protein.

Isoelectricfocusing In isoelectric focusing, proteins are separated by electrophoresis in a pH

gradient in a gel. They separate on the basis of their relative content of
positively and negatively charged residues. Each protein migrates through the
gel until it reaches the point where it has no net charge, its isoelectric point (pl).

isualzation of Proteins can be visualized directly in gels by staining them with the dye
Proteins in gels Coomassie brilliant blue or with a silver stain. Radioactively labeled proteins
can be detected by overlaying the gel with X-ray film and observing the
darkened areas on the developed autoradiograph that correspond to the
radiolabeled proteins. A specific protein of. interest can be detected by
immunoblot (Western blot) following its transfer from the gel to nitrocellulose
using an antibody that specifically recognizes it. This primary antibody is then
detected with either a radiolabeled or enzyme-linked secondary antibody

Related topics Protein structure (B3) Antibodies as tools (D5)

Protein purification (B6)

Native PAGE When placed in an electric field, molecules with a net charge, such as proteins
SDS-F
will move towards one electrode or the other, a phenomenon known as elec-
trophoresis. The greater the net charge the faster the molecule will move. In
native polyacrylamide gel electrophoresis (PAGE) the molecular separation is
based on the size of the protein as well as its net charge since the electrophoretic
separation is carried out in a gel which serves as a molecular sieve. Small mole-
cules move readily through the pores in the gel, whereas larger molecules are
retarded. The gels are commonly made of polyacrylamide which is chemicall
 inert and which is readily formed by the polymerization of acrylamide. The poseo
sizes in the gel can be-controlled by choosing appropriate concentrations of acryl
amide and the cross-linking reagent, methylene bisacrylamide. The higher the
çoncentration of acrylamide used, the smaller the pore size in the final gel. The
8el is usually cast between two glass plates of 7-20 cm separated by a distance of
0.5-1.0 mm. The protein sample is added to wells in the top of the gel, which are
formed by placing a plastic comb in the gel solution before it sets (Fig. 1). Ablue
dye (bromopheno! blue) is mixed with the protein sample toaid its loading on to
the gel. Because bromophenol blue is a small molecule, it also migrates quickly
e applied to a
through the gel during electrophoresis and so indicates the progress of elec-
n the basis of trophoresis. In the simplest form of native PAGE, the buffer, which is the same in
both the upper and lower reservoirs and in the gel, has a pH of approximately9,
ively charged such that most proteins have net negative charges and will migrate towards the
tively charged
anode in the lower reservoir. An electric current (approximately 300 V) is applied
across the gel from top to bottom for 30-90 min in order to move the proteins
gent to break through the gel. The gel is then removed from the electrophoresis apparatus, and
odecyl sulfate the proteins within it visualized.
erall negative
s through a
harge to mass Sample wells Cathode
oroteins move
Upper buffer
y of a protein
reservoir
polypeptide
LLUL
esis in a pH Sample
e content of
s through the Polyacrylamide gel
sandwiched
ric point (p). between two
glass plates
vith the dye Anode
-eled proteins
bserving the
pond to the Lower buffer
reservoir
detected by
nitrocellulose
ibody is then
Fig. 1. Native polyacrylamide gel electrophoresis. The protein samples are loaded into the
ntibody. sample wells formed in the top of the gel. An electric field is applied across the gel from top
to bottom and the proteins migrate down through the gel. The smaller the protein and the
greater its net negative charge, the further it will migrate.

In SDS-PAGE, the proteins are denatured and coated with an overall negative
SDS-PAGE charge [due to bound sodium dodecyl sulfate (SDS) molecules] and thus the
ich as proteins,
known as elec- basis for their separation is only their mass. The protein mixture is first treated
will move. In with à reducing agent such as 2-mercaptoethanol or dithiothreitol to break all the
r separation is disulfide bonds (see Topic B3). The strong anionic detergent SDS is then
electrophoretic added which disrupts nearly all the noncovalent interactions in the protein,
we. Small mole- unfolding the polypeptide chain. Approximately one molecule of SDS binds via
molecules are its hydrophobic alkyl chain to the polypeptide backbone for every two amino
h is chemically acid residues, which gives the denatured protein a large net negative charge that
is proportional to its mass. The SDS/protein mixture is then applied to sample
 Section B Amino acids and protein 8 Electr

(a) (b)

94
43
30

14.4
Distance migrated
Fig. 2. SDS-PAGE. (a) Appearance of proteins after electrophoresis on an SDS polyacrylamide gel.
Lane 1, proteins (markers) of known molecular mass; lane 2, unpurified mixture of proteins; lane 3,
detemination of the
partialy purified protein; lane 4, protein purified to apparent homogeneity; (b)
molecular mass of an unknown protein by comparison of its electrophoretic mobility (distance
migrated) with those of proteins (markers) of known molecular mass.

wells in the top of a polyacrylamide gel as in native PAGE (see Fig. 1). After
carrying out electrophoresis, the gel is removed from the apparatus and the
proteins visualized (Fig. 2a). Small proteins move furthest through the gel, where-
as large ones move more slowly as they are held back by the cross-linking in the
gel and remain near the top. Under these conuiions, the mobility of most
polypeptide chains is linearly proportional to the logarithm of their mass. Thus,i
if proteins of known molecular mass are electrophoresed alongside the samples,
the mass of the unknown proteins can be determined (Fig. 26). Proteins that difer
in mass by about 2% (e.g. 40 and 41 kDa; a difference of approximately 10 amino
acid residues) can usually be distinguished. SDS-PAGE is a rapid, sensitive and
widely used technique from which can be determined the degree of purity of a
protein sample, the molecular mass of an unknown protein and the number of

polypeptide subunits within a protein (see Topic B3).

Isoelectric focusing electrophoretically separates proteins on the basis of their
Isoelectric
relative content of positively and negatively charged groups. When a proteinj
focusing
is at its pl (see Topic B2), its net charge is zero and hence it will not move ing
an electric field.
In isoelectric focusing, a polyacrylamide gel is used which has large pores (so
as not to impede protein migration) and contains a mixture of polyampholytes
(small multi-charged polymers that have many pl values). If an electric field is
applied to the gel, the polyampholytes migrate and produce a pH gradient. To
separate proteins by isoelectric focusing, they are electrophoresed through such a
gel. Each protein will migrate through the gel until it reaches a position at which
the pH is equal to its pl (Fig. 3). If a protein diffuses away from this position, its
net charge will change as it moves into a region of different pH and the resultingisualizati
In this way eachproteins in
electrophoretic forces will move it back to its isoelectric position.
protein is focused into a narrow band (as thin as 0.01 pH unit) about its pk
Isoelectric focusing can be combined with SDS-PAGE to obtain very high reso0
lution separations in a procedure known as two-dimensional gel electrophoresis.|
The protein sample is first subjected to isoelectric focusing in a narrow strip off
gel containing polyampholytes. This gel strip is then placed on top of an SDS
polyacrylamide gel and electrophoresed to produce a two-dimensional pattern of
spots in which the proteins have been separated in the horizontal direction on the
basis of their pl, and in the vertical direction on the basis of their mass (Fig. 4). The
overall result is that proteins are separated both on the basis of their size and their|
ids and protel Electrophoresis of proteins

(b)

7.0 7.0

6.0
6.0

5.0 5.0
olyacrylamide gel
oroteins; lane 3,
mination of the O
v (distance
Fig.3. soelectric focusing. (a) Before applying an electric curent. (o) after appying an
the
electric cument proteins migrate to a position at which their net charge is zero
(isoelectric point, pl).
see Fig. 1). Afte
paratus and the
h the gel, where
Dss-linking in the 1) Isoelectric focusing9
nobility of most pH 10 pH 4
heir mass. Thus
Protein bands
side the samples
oteins that diffe
mately 10 amino
ia, sensitive and sDS gel
electrophoresis
ree of purity of a
d the number of
Protein spots

ne basis of their
When a protein
will not move in Fig. 4. Two-dimensional gel electrophoresis. The protein
sample is first subjected to
isoelectric focusing in one dimension and then to SDS-PAGE in the second dimension.

as large pores (so charge. Thus two proteins that have very similar identical pls, and produce
polyampholytest single band by isoelectric focusing, will produce
or
a
n electric field is gel electrophoresis two spots by two-dimensional
(see Fig. 4). Similarly, proteins with similar
pH gradient. To or identical molec
d through such at
ular masses, which would produce a single band by SDS-PAGE, also produce two
osition at which/
spots because of the initial separation by isoelectric focusing.
this position, its
and the resultingisualization of As most proteins are not
directly visible on gels to the naked eye, a method
In this way eaciproteins
in gels has to be employed in order to visualize them
bout its pk commonly used protein stain is the dye Coomassie
following electrophoresis. The most
brilliant blue. After electro-
n very high reso phoresis, the gel containing the. separated proteins is
immersed in an acidic
electrophoresis alcoholic solution of the dye. This denatures the
proteins, fixes them in the gel so
anarrow strip o that they do not wash out, and allows the
dye to bind to them. After washing away
n
top of an SDS excess dye, the proteins are visible as
discrete blue bands (see Fig. 2a). As little as
sional pattern of 0.1-1.0 Hg of a protein in a gel can be visualized
l direction using Coomassie brilliant blue. A
on the more sensitive general
protein stain involves soaking the gel in a silver salt
mass (Fig. 4). The solution. However, this technique is rather more difficult to If the
eir size and their apply.
sample is radioactive the proteins can be visualized indirectly protein
by overlaying the
 Section B- Amino acids and
protet. Sect

gel with sheet of X-ray film. With time (hours to weeks

a

radioactivity of the sample proteins), the radiation emitted willdepending

cause a
on t BS
of the film. Upon
development of the film the resulting darkenir
darkened areas corresponding to the autoradiograph will hav
Another way of visualizing the
positions of the radiolabeled proteins.
protein of interest is to use an antibody againg
the protein in an immuhoblot (Western blot) (see
the proteins have to be transferred out of the
Topic D5). For this technique
gel on to a sheet of nitrocellulose
nylon membrane. This is accomplished by overlaying the
gel with the nitrocelle
lose and blotting the protein on to it
by applying an electric current. The nitro
cellulose then has an exact image of the pattern of proteins that was in the
excess binding sites on the nitrocellulose are then blocked with a
gel. Th
tein solution such as milk powder, before
nonspecific pr
placing the nitrocellulose in a solution
containing the antibody that recognizes the protein of interest (the primary ant
body). After removing excess unbound antibody, the primary antibody that
now
specifically bound to the protein of interest is detected with either a radio
labeled or enzyme-coupled secondary
antibody. Finally,the secondary antibod
is detected either by
placing nitrocellulose against a sheet of X-ray film (if
the
radiolabeled secondary antibody has been used), or by
a solution of a substrate that is converted into
adding to the nitrocellose
colored insoluble product by the
a
nzyme that is coupled to the secondary antibody.