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Purification
by
Column
Chromatography
Proteins
play
key
roles
in
various
macromolecular
functions
in
all
living
organisms.
Catalyzing
metabolic
processes,
acting
as
structural
elements,
conveying
intra-
and
extracellular
signals
are
only
three
of
the
many
functions
they
play
in
the
body.
To
understand
their
exact
roles
and
functions
we
must
isolate
the
proteins
and
analyze
them
using
some
of
the
most
modern
biochemistry
procedures.
Column
chromatography
takes
into
account
the
affinity
a
protein
will
have
to
specific
solutions
due
to
their
chemical
properties.
In
this
instructional
set,
water
and
Hydroxycitric
acid
(5%
HCA)
are
used
to
wash
away
impurities
from
the
column
while
pure
Methanol
(MeOH)
will
be
used
to
elute
the
columns
to
obtain
purified
proteins.
Note:
There
is
no
single
way
of
purifying
protein,
this
is
just
one
of
the
possible
purification
methods.
Materials
1. Chromatography
apparatus
2. Vacuum
pump
3. Columns
for
protein
purification
4. Pipette
&
Pipette
tips
5. 2ml
Vials
6. 100%
MeOH
7. 5%
HCA
8. Your
protein
solutions
9. Paper
towels
10. Nitrile
Gloves
Columns
for
11. Distilled
water
protein
purification
Chromatography apparatus
Vacuum Pump
Instructions
Warning:
Wear
laboratory
gloves
and
coat
during
entire
procedure
to
prevent
any
injuries
when
handling
the
solutions
Preparation
1. Obtain
your
protein
samples1
and
place
near
the
chromatography
apparatus
on
your
work
bench
2. Label
a
second
set
of
clean
2mL
vials,
as
show
in
the
example
below.
Be
sure
to
include
at
least:
a. Sample
name
b. Name
of
the
experiment
3. Place
your
container
of
5%
HCA,
your
container
of
pure
Methanol
(MeOH)
and
a
bottle
of
distilled
water
on
your
bench
4. Set
a
pipette
to
3ml,
put
a
pipette
tip
on
it
and
place
it
near
the
5%
HCA
5. Calibrate
a
second
pipetted
to
4ml,
put
a
tip
on
it
and
place
near
the
MeOH
6. Set
out
a
few
paper
towels
near
the
chromatography
machine
7. Place
chromatography
columns
near
the
chromatography
machine
Now,
you
are
ready
to
run
your
samples
and
purify
your
proteins.
Tips
to
consider
before
running
the
chromatography:
o Between
steps
6
and
7:
Do
not
let
the
columns
run
dry
when
adding
the
HCA
after
the
addition
of
MeOH
o Achieve
this
by
opening
and
running
the
columns
one
at
a
time
o In
step
8:
When
loading
your
protein
samples,
be
sure
to
keep
track
of
which
sample
is
in
which
column
o In
step
11:
Add
distilled
water
when
about
10-15
mm
of
protein
sample
remains
in
the
column
o For
step
14:
Be
sure
to
place
the
correct
vial
under
the
appropriate
collection
spout
1
Crude
protein
samples
are
to
be
pre-prepared
in
vials
and
suspended
in
appropriate
buffer
solution
3. Turn
vacuum
pump
on
4. Open
turn
the
pressure
knob
where
the
vacuum
tube
is
connected
to
the
chromatography
apparatus,
this
is
allow
pressure
to
build
in
the
chromatography
chamber2
a. Allow
pressure
to
increase
to
20-25mmHg
The
pressure
knob
5. Add
3mL
of
MeOH
to
each
column
6. Turn
open
the
spout
of
each
column,
so
that
the
MeOH
in
each
column
runs
through
the
column
and
into
the
chamber
Note:
Do
not
let
the
column
run
dry
before
putting
in
the
5%
HCA
7. Pipette
3mL
of
5%
HCA
into
each
column
a. Run
each
column
dry
and
close
instantly
8. Load
your
protein
samples
to
each
column
9. Lower
the
pressure
to
5-10
mmHg
10. Open
every
column
and
allow
the
samples
to
run
through3
11. Before
the
columns
run
dry,
add
1-2
mL
of
distilled
water,
this
will
wash
out
any
remaining
compounds
from
the
column
12. Turn
vacuum
off
and
release
any
pressure
left
in
the
chamber
13. Clean
out
the
inside
of
the
chamber
with
a
paper
towel4
14. Load
your
clean
and
labeled
vials
to
the
inside
of
the
chamber
15. Place
the
top
of
the
chromatography
chamber
back
onto
the
apparatus
16. Bring
inner-chamber
pressure
back
up
to
20-25
mmHg
2
Pressure
in
the
chamber
is
measure
in
mmHg
3
All
columns
can
be
open
at
the
same
time
since
pressure
is
so
low,
you
will
be
less
time
pressured
4
This
will
prevent
any
of
the
remaining
MeOH
to
wash
away
the
labels
on
your
vials
Release
the
chamber
pressure,
open
the
apparatus
and
remove
your
vials
containing
your
purified
protein
samples!