Chromatography
from Greek χρῶμα chroma "color" and
γράφειν graphein "to write".
Background
!
!
http://en.wikipedia.org/wiki/Chromatography (12 Jan 2013)
General chromatography:
text book experiment 2, beginning on page 25
Affinity chromatography: experiment #10, pages 157-162.
Chromatography is a technique that allows for
the separation of molecules based on their
differential migration through a porous medium.
Molecules partition between the stationary phase
and the mobile phase, and this ‘relative mobility’
determines the relative rate of migration.
!
Rf = (dist. traveled by sample)/(dist. to solv. front)
!
The closer Rf is to 1, the faster the migration
!
The closer Rf is to 0, the slower the migration
!
Rf= 1-α.
spinach leave pigments
α=
Mobile phase = nail polish remover
(acetone, ethyl acetate, butyl acetate),
stationary phase = coffee filter paper.
•
In 9:1 pet ether: acetone
•
migration
carotene Rf = 0.98
xanthophyll Rf = 0.90
chlorophyll a Rf = 0.83
chlorophyll b Rf = 0.71
•
http://www.layers-of-learning.com/why-do-leaves-change-color-in-the-fall/
2
α is the partition coefficient
[ ] in stationary phase
[ ] in stationary and mobile phase
In column chromatography, the stationary phase
is a resin.
The mobile phase is a buffer solution that flows
over the resin carrying analyte with it. (More
generally, the mobile phase can be mixtures of
solvents other than water. Even biomolecules
may be separated in water:acetone and
water:acetonitrile mixtures.
The analyte is characterized by the relative
extents to which it interacts with the mobile vs.
the stationary phases.
many types of chromatography
Chromatography video.
!
!
!
exploit different interactions.
For purposes of separation, exploit a
feature that distinguishes different
compounds present in a mixture, such as
size, charge, polarity ...
For the example of plant pigments the
solid phase is cellulose (HCOH)n and the
mobile phase is 10% H3COCH3 and 90%
light hydrocarbons (pentane-heptane).
Absorbance
Rf = 0.98
Rf = 0.90
elution volume
Rf = 0.83
3
http://www.youtube.com/watch?v=9GiLjH9Oym8
4
Rf = 0.71
Example: plant pigments (again)
Chromatography of many
kinds
Rf = 0.98
Rf = 0.90
Rf = 0.83
See tables 2-1, 2-2 in text book
!
Gel filtration: retardation of large
molecules
!
Ion-exchange: electrostatic retention
of molecules with a charge opposite to
that of the resin.
!
Hydrophobic interaction: non-polar
surface groups cause molecule to be
retained on column (also called
reverse phase).
!
Affinity: exploits a specific affinity of
the molecule for a ligand that can be
built into the column or attached to it.
(Also requires a release mechanism).
Rf = 0.71
carotene Rf = 0.98
xanthophyll Rf = 0.90
chlorophyll a Rf = 0.83
chlorophyll b Rf = 0.71
migration
On cellulose (filter paper)
In 9:1 pet ether: acetone
5
!
http://www.layers-of-learning.com/why-do-leaves-change-color-in-the-fall/
6
Different resins:
Size-Exclusion
!
!
Relies on the ability of a given sized molecule
to enter the uniformly sized pores of a solid
matrix. Molecules that are too big are
excluded from the mobile phase within the
pores, and exit the matrix faster. Smaller
molecules have to go through a larger volume
of mobile phase, and so exit later.
The volume accessible to the molecule
dictates its rate of migration.
Ion-Exchange
•
Relies on the differential electrostatic affinities
of molecules carrying a surface charge for an
inert, charged stationary phase.
•
•
•
Cation exchange has negative stationary phase
• Molecules with positive charge will interact with resin.
Anion exchange has a positive stationary phase
• Molecules with negative charge will interact with resin.
Molecules are eluted by either changing the pH
or the ionic strength
•
•
pH: the molecule is adsorbed to the column at a
pH that gives it a charge opposite that of the
resin, and is eluted by a change in pH that
causes it to lose its charge or affinity for the resin.
Ionic strength: as the concentration of counterions in the mobile phase increases, they
electrostatically compete with adsorbed
molecules bound to the charged resin. Molecules
with lower net charge elute with lower salt
concentrations.
Ion-exchange
chromatography
Ion-Exchange
!
Carboxymethyl (CM) cellulose is a cation exchanger
http://www.genome.ou.edu/3653/Lecture12-9_18_06
!Carboxymethyl
(CM) cellulose is a cation exchanger
(DEAE) cellulose is an anion exchanger
!Diethylaminoethyl
!Elute
by increasing ionic strength or by changing pH
(chromatofocussing).
Blue dextran MW = 2,000,000 neutral
cytochrome C MW = 12,000 pI = 10.7
DNP-glycine MW = 241 pI = 3
In ion-exchange mode which should elute first ?
In gel filtration mode which should elute first ?
10
resolution on CM sephadex
Resolution:
separation vs. spreading
!
Affinity Chromatography
27 mimics a ligand of the protein.
Use a resin that
Eg. blue columns in which the resin is
velopment of the column. Many chromatographic
decorated
with EXPERIMENT
molecules
of cibacron 37
blue.
columns, however, are eluted with
some sort of mo2
Chromatography
EXPERIMENT 2
R = 2d /(W1+ W2)
Concentration of compounds
d
Chromatography
bile phase gradient. Here, the nature of the mobile
phase
changes continuously
“activate”
carbohydrate-based
resins for throughout
derivatiza- develop- O
NH2
ment
with
respect
to
ionic
strength,
pH,
tion with a ligand of interest (Fig. 2-6). Most
ofligand conSO!
3
centration,
or organic:aqueous
ratio. As one
these chemical
reactions
take place on thesolvent
hydroxyl
H
A
B
or the
more
of and
thesecreate
parameters
changes,
groups of
resin
intermediates
thatso too does
N
SO!
3
H
the to
partition
coefficient
of one
oramino
more molecules
are subject
nucleophilic
attack by
a free
N
group on
the desired
adsorbed
on ligand.
the stationary phase. Although this O
N
H
W2
greatly complicates the calculation of $, it can
N
Dye-Ligand
Chromatography.
Of
all
the
types
of
greatly enhance the ability of a given sized column
N
N
chromatography
presented
in thisofchapter,
the prinW1
to separate
a number
different
compounds. By
SO!
3
Cl
ciples governing
the
technique
of
dye-ligand
chrocontinuously changing the partition coefficients of
matography
are
perhaps
the
least
well
understood.
Elution volume
molecules passing through the stationary phase,
you
Figure 2-7 Structure of Cibacron Blue F3GA.
Purely by accident in the 1960s, a blue dye known
can effectively increase the number of plate transas Cibacron Blue was found to have strong affinity
Figure 2-2 Resolution of two compounds (A and
occurring
a given
lengthkinase
of the column.
for two fers
enzymes
foundover
in yeast:
pyruvate
B) emerging from a chromatography
A
20-ml
column
that
provides
10
plate
transfers utiand phosphofructokinase. As shown in Fig. 2-7,
column.
lizing
an
isocratic
elution
scheme
may
100 or ligands commonly used to elute proteins
strates
Cibacron Blue consists of a series of individual andprovide
to
200
plate
transfers
when
used
in
conjunction
with
from Cibacron Blue columns include: S-adenosylaromatic rings containing several conjugated
! High-α analytes tend to have more fused
zone
an elution
gradient.
methionine (SAM), ATP, GTP, NAD$, NADH,
double bonds
common
to molecules that absorb
light inthe
the visible
range of
wavelengths.
Ringencountered
strucADP,
spreading (continuously released from
Remember
that
one problem
withAMP, and cAMP. If a suitable ligand cannot
areas of high concentration to areas of low contures similar
to this
presentofinplate
purine
nu- is an
be identified
that will cause the protein of interest
increasing
theare
number
transfers
instationary
into
mobile
phase
passing
centration asphase
the mobile
phase
continuously
passes
cleotides.
This inmolecule
also contains
three sulto elute from the column, elution can be achieved
crease
zone spreading,
decreasing
the resolution
!
fonic acid
groups peaks
that may
be mistaken
for
using
by).through the stationary phase.
of (SO
two3!)elution
containing
compounds
withthe same mobile-phase gradients commonly
phosphate
(PO
)
groups
by
an
enzyme.
Based
on
used
with cation-exchange columns: increasing pH
4
similar partition coefficients. Gradient elution
! A long
these two
pieces
of
evidence,
it
is
currently
believed
or
ionic
column
notof the
Resolution.
The is
goal
any solution.
chromatographic
schemes often work to minimize zone spreading. strength. Two commercially available dyethat Cibacron Blue acts as a substrate analog of
ligand chromatography adsorbents are shown in
step is to maximize resolution or to minimize zone
Recall that molecules with lower $ values show less
! Employ
ADP-ribose,
explaining
why
the
molecule
has
been
Table 2-1.
gradient
elution
instead
of
spreading. Resolution is a function of the position
zone spreading after a given number of plate transfound to have affinity for many different NAD!of the maximumisocratic
elution peak resolution.
height and the elu(unchanging)
fers
than molecules
with large
values. If a Immunoaffinity
molerequiring
enzymes
(dehydrogenases)
and$ kinases
+ Chromatography. As will be detion peak width (Fig. 2-2). The greater the resoluculebind
thatpurine
has a relatively
large $ value underscribed
load- in Section IV, an antibody is capable of se(commonly
nucleotides).
tion between two elution peaks, the greater the de- Unfortunately,
ing conditions
can be eluted
gradientlectively
that recognizing and binding a single protein
the specificity
of thiswith
blue adye
gree of separation between the two molecules.
decreases
$ value isasnot
it passes
for the continually
types of enzymes
just its
described
as through
or other antigen in the presence of thousands of
Mathematically, resolution (R) is two times the discolumn,
zone spreading
can be minimized,
andproteins and other biological molecules. Natgreat as the
originally
thought.
Indeed, Cibacron
Blue
other
tance between two elution peak maxima (d) divided
has beenresolution
proven to between
have greatneighboring
affinity for aelution
num- peaks
urally,
can then, immunoaffinity chromatography offers
by the sum of the widths of the two elution peaks
ber of seemingly
unrelated
proteins,mobile
such as
!- gradients
perhaps the greatest potential in attempting to pube maintained.
In general,
phase
interferon
blood
serum
albumin.
The nonspea protein in a single step. A purified antibody
(W1 ! W2):
areand
most
often
used
to optimize
a specific rify
chro-
11
This is mis-recognized as a substrate analog
by enzymes that bind NADH and purine
nucleotides.
Such enzymes bind to the resin more strongly
than random proteins do. The latter can be
washed away and then the proteins binding
the resin can be eluted with authentic
substrates, cofactors or analogs (eg. SAM,
ATP, GTP, NAD , NADH, FMN ...
Ni2+-affinity chromatography
Ni2+ column IMAC
EXPERIMENT 2
!
General protocol involves three steps
–
–
–
!
!
!
13
Bind interesting protein to
chromatography resin.
Wash away all uninteresting proteins.
Elute interesting protein from
chromatography resin (also called
matrix).
We will use a ‘batch’ method rather
than a chromatographic application.
Lower resolution but much faster.
Depends on ability to find conditions
where the target protein binds
completely and everything else does
not. Modern molecular biology
makes this easy ...
Chromatography
O
CH2
CH
C
O
O!
Ni2!
CH2
O
C
O!
Ni2!
C
O!
N
OH
Solid
support
CH2
CH
CH2
Figure 2-8
Solid
support
CH2
CH
CH2
C
O
O!
Fe3!
CH2
O
C
O!
Fe3!
C
O!
N
CH
CH2
Protein
Ni2!
O
OH
6 ! His
Phosphoprotein
Fe3!
Figure 2-8 Immobilized metal affinity chromatography resin (nitrilotriacetic acid).
some cases, these types of adsorbents can be debile phase. Compounds will display different rates
rivatized with DEAE, polyethyleneimine, acetyl, or
of migration through the stationary phase dependC18 groups.
ing on their polarity. More-polar compounds will
In normal-phase partition chromatography, the
have more affinity for the polar stationary phase
stationary phase is polar and the mobile phase used
than for2+
the nonpolar mobile phase. As a result, the
Onlyisproteins
that
can tightly
to Ni will
be preferentially
in development
nonpolar. In
reverse-phase
par-bindmore-polar
compounds
will travel less distance
retained on
column.
tition chromatography,
thethe
stationary
phase is nonacross the plate than the nonpolar compounds,
polar and Such
the mobile
phase used
in rare
development
is
which
have a higher
affinity for the
proteins
are
in nature,
so
a protein
engineered
to nonpolar mopolar. In thin-layer partition chromatography, the
bile phase. The opposite is true for reverse-phase
have this capability is generally unique,
and the only protein
samples are spotted in a straight line across the botpartition chromatography: the more polar the combesupport
retained
onorigin).
suchCare
a column.
tom of theto
solid
(at the
must
pound, the greater its affinity for the mobile phase,
be taken to avoid widespread diffusion of these samthe further that compound will travel across the
ples (small aliquots of the total applied sample are
plate in a given period of time. The behavior of a
spotted and allowed to dry). After all the applied
compound in thin-layer partition chromatography
samples have dried, the plate is placed, origin side
is usually described in terms of relative mobility
down, in a chamber containing a small amount of
(Rf ):
the mobile-phase solvent on the bottom. It is esDistance traveled by sample (cm)
sential that the origin lies above the level of the
Rf " #####
mobile-phase solvent in the chamber (see Fig. 6-8)
Distance traveled by solvent front (cm)
and that the chamber is saturated with the vapors
of the mobile phase before development is begun.
The larger the value of Rf, the more affinity the
The mobile phases used in the development of
compound has for a particular mobile phase solthin-layer chromatography plates are usually comvent.
14
3
Native conditions
lysate
flow-through,
eluate
Cell lysate
Denaturing conditions
lysate
flow-through,
eluate
Very clean samples with
few steps
Qiagen manual “Ni-NTAspin.pdf”
Ni-NTA
spin column
Bind
Wash
llama
shark
Elute
Pure 6xHis-tagged protein
Figure 1. Ni-NTA Spin purification procedure.
15
16
6
Ni-NTA Spin Handbook 01/2000
Single-domain antibodies: promising experimental and
therapeutic tools in infection and immunity. Wesolowski et al
(2009) Med. Micro. Immun. 198(3).
Encoding
affinity for Ni2+
EcoRI (5740)
AatII (5669)*
NheI (32)*
EcoRV (192)
T7 term
T7 reverse
XhoI (328)*
AccI (344)
SacI (354)*
BamHI (359)*
EcoRI (368)
NdeI (377)*
TEV
His
NcoI (423)*
XbaI (462)*
T7
T7 forward
BglII (528)*
AmpR
PstI (4992)*
Electrophoresis through 12%
acrylamide, stained with
Coomassie brilliant blue
rbs
SphI (725)*
pBR322 origin
pET15TEV_NESG
5741 bp
ApaI (1461)*
BssHII (1661)*
EcoRV (1700)
migration
BsaI (4808)*
BsmBI (1865)
TEV
M G H H H H H H E N LY F Q S H M G SAN SAD PAS S V DAK L L
2
Normal protein seq.
18
www.chembio.uoguelph.ca/educmat/chm357/affinity.pdf
pure GSTfusion
3 hr. IPTG
2 hr. IPTG
1 hr. IPTG
Pre-IPTG
MW stdds
Init
Sal I
XhoI
Nco I|
Nde I
EcoR I
BamH I
Sac I"
HinD III
|
|
TEV Protease
|
|
|
|
|
|
|
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC
______6XHis tag____
4 hr. IPTG
AccI (3624)
BsmBI (3495)
The experiment
What you will do:
Day 1. Use Ni-resin to purify a
flavoenzyme (yellow-coloured).
Day 2. Use SDS-PAGE to visualize
the proteins present in different
fractions of the column purification,
see text experiment 4.
Chromatographic fractions to save and characterize by SDS
PAGE:
Cells prior to induction with IPTG*.
Cells after induction with IPTG*.
Cleared extract (lysed cells minus debris)
Column flow-through
Column wash
1st eluate
Rainbow Low Molecular Weight markers
* provided by T.A.
19
Producing the protein extract
(this will be done for you, but include it in
your notebook)
Grow E. coli bearing expression plasmid to
OD600 ≈ 0.5
! Collect a ‘pre-IPTG’ sample by centrifugation.
! Add IPTG to initiate expression.
! Grow 3 hr for protein production.
! Harvest by centrifugation, discard supernatant.
! Resuspend cell pellet in lysis buffer, centrifuge
down cells again.
! Retain a second sample (‘post IPTG’).
! Resuspend again in lysis buffer, add lysozyme
(1mg/ml), incubate 30 min.
! Sonicate in bursts to minimize heating.
! Centrifuge out debris at 10,000 x g for 20-30
Buffers
for purification under native conditions (from E. coli and insect cells; protocols 6,
min.
!
9, 11, 12, 13, 14, and 16)
Lysis buffer (1 liter):
prevents non-specific binding to Ni-resin.
50 mM NaH2PO4
6.90 g NaH2PO4·H2O (MW 137.99 g/mol)
300 mM NaCl
17.54 g NaCl (MW 58.44 g/mol)
10 mM imidazole
0.68 g imidazole (MW 68.08 g/mol)
20 Adjust pH to 8.0 using NaOH.
Wash buffer (1 liter):
50 mM NaH2PO4
6.90 g NaH2PO4·H2O (MW 137.99 g/mol)
300 mM NaCl
17.54 g NaCl (MW 58.44 g/mol)
20 mM imidazole
1.36 g imidazole (MW 68.08 g/mol)
How you will do it:
1. Running the Ni column (from
the QIAgen manual)
3.
1.
Translation: add 600 µl NPI-10 buffer to the top
compartment of your spin column and then spin for 2
min (T.A. will determine the speed to be used). Discard
solution that passes into the lower compartment of the
spin column, the ‘flow-through’.
Translation: add 600 µl NPI-20 buffer to the top
compartment of your spin column and then spin for 2
min (T.A. will determine the speed to be used). Save
flow-through, this is the sample called ‘column wash’.
4.
2.
Translation: add 300 µl NPI-500 buffer to the top
compartment of your spin column and then spin for 2
min (T.A. will determine the speed to be used). Save
flow-through, which is now called the 1st eluate.
Add a second 300 µl of NPI-500 buffer to the top
compartment, spin again and save the flow-though as
2nd eluate.
Compare the colours of the 1st and 2nd eluate. You
will make an SDS-PAGE sample of the 1st eluate.
Translation: Deliver 600 µl of the cleared cell lysate
into the top compartment of your spin column and then
spin for 5 min (T.A. will determine the speed to be
used). Save flow-through, you will run an SDS-PAGE
sample of this, called ‘flow-through’.
Also see:
21
22
SDS PAGE
(see experiment 4 in text)
2. Samples to collect for
SDS PAGE
!
!
Draw arrows into your flow chart showing when these fractions
are collected and set aside
Cells prior to induction with IPTG*.
Cells after induction with IPTG*.
Cleared lysate
Column flow-through
Column wash
1st Eluate
!
!
* provided by T.A.
!
Fractions will be treated with SDS-PAGE sample buffer,
denatured by incubation at 90 °C, and run on a
polyacrylamide† gel along with molecular weight standards, on
day 2.
†
polyacrylamide is a neurotoxin.
!
23
24
!
The text describes the principles and
apparatus.
We will use purchased (precast) gels.
You will simply need to add samples into
the wells, add buffers, hook up to power
and supervise.
Gels will be removed from glass plates
and stained with our old friend
Coomassie brilliant blue.
All materials will be made for you. Your
execution plan simply needs to provide a
sketch of your planned loading
arrangement (what samples in which
lanes).
We WILL do the data analysis steps 1-4
of using the mobilities of molecular
weight standards to determine the
molecular weight of our test protein (text
pg 76).
(see pages 74-77)