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A project funded by the California Community Chancellor’s Office
to serve the employment and educational needs of the biotechnology community
of Southern California
Easy Classroom Column Chromatography
Laboratory Activity from the
Southern California Biotechnology
Center.
For more information, please contact
us at (760) 795-6648
Purpose:
To demonstrate the principle
and practice of size exclusion column chromatography, a technique that
is routinely used in the separation and purification of proteins. Students
will see how columns are used to separate biomolecules from one another
based on their size. Three colored proteins (blue dextran, cytochrome c,
and vitamin B12) will be separated from each other based on differences
in their molecular weights during this activity.
Materials:
(If you reside within
our service area, materials and training to assist you to perform the procedure
are available from the Southern California Biotechnology Center)
-
Biogel P20 or P30 chromatography resin
[1 g resin per 10 ml column]
-
Colored protein standards mix [blue dextran,
cytochrome c and vitamin B12, at 2.5 mg per ml each]
-
NAT buffer [200 mM NaCl, 50 mM Tris, pH
9]
-
Disposable glass or plastic 10 ml per pipette
with top "restricted diameter" portion removed
-
Glass wool
-
Tubing
-
Clamp of some sort to stop liquid flow
through tubing
-
Pasteur pipettes or other transfer device
to place resin slurry in column and to apply protein mix to column.
-
Tubes to collect eluted proteins from column
-
Ring stand or other apparatus to hold column
upright and vertical.
Southern California Biotechnology
Center
(760) 795-6648
http://www.miracosta.cc.ca.us/scbc
scbc@mcc.miracosta.cc.ca.us
One Barnard Drive, Oceanside, CA 92056-3899
Background
Principles of Size Exclusion
Chromatography [taken from Biotechnology: Proteins to PCR by Burden
and Whitney (1995) Birkhauser, publisher]
Gel filtration (GF) chromatography
separates globular proteins by size (i.e., molecular weight) and consists
of a column packed with porous polymeric beads. These beads are usually
composed of cross-linked polysaccharides (some GF media use synthetic polymers
such as polyacrylamide) in which the pore sizes within the gel beads are
controlled.
A sample is applied to the
top of a column filled with gel, and the protein is eluted by passing a
defined volume of a suitable buffer through the gel matrix. The larger
proteins cannot physically enter the small pores of the beads, and, therefore,
spend less time in the gel bead and more time in the eluting buffer. The
eluting buffer is sometimes referred to as the mobile phase, and the gel
itself is called the stationary phase. Each bead is similar to a three
dimensional maze that retards the elution of the smaller molecules. As
such, the larger molecules elute from the column first, followed by the
smaller molecules, and the result is the fractionation of the protein mixture
by size. The size of a globular protein is related to its molecular weight
so gel filtration effectively separates proteins by the differences in
their molecular weight (Figure 5.4).
In this way, gel filtration
chromatography can be used to estimate the molecular weight of a protein.
The retention times (or volumes) of a series of globular proteins with
known molecular weights are determined. The data are used to plot log molecular
weight versus retention time to generate a calibration curve that can be
used to estimate the molecular weight of an unknown protein. The linear
relationship breaks down with nonglobular proteins and DNA since both are
nonspherical in shape.
The resolution in gel filtration
is dependent on column length, the flow rate of elution, pore size of the
gel, and the particle size of the gel bead. In general, the efficiency,
and therefore the resolution, of any chromatographic analysis improves
as the size of the gel bead decreases. The pore size of the gel determines
the molecular weight range over which separation will occur. The column
length is important in gel filtration, and resolution generally increases
with increasing column length. However, increasing column length eventually
degrades resolution due to diffusion of the sample as it transverses the
gel filtration column. In addition to good recovery and mild conditions,
gel filtration usually gives good resolution and is relatively easy to
perform.
Teacher Preparation:
Day before activity:
Preparing the Biogel resin for use:
Hydrate the Biogel resin by adding 40
ml NAT buffer to 1 gram of resin. Store overnight in the cold (refrigerated).
You will need 1 g resin for each column you prepare.
The day of activity:
"Define" the Biogel resin before use by
performing the following, see figure below (a);
-
Swirl the hydrated Biogel slurry and allow
the resin to settle for several minutes by gravity.
-
Decant or aspirate off the supernatant and
discard it.
-
Add cold, fresh NAT buffer (approximately
40 ml) per gram of resin, swirl and allow the resin to settle as above.
-
Repeat the decanting step as above.
-
Add approximately 25 ml cold, fresh NAT buffer
per gram of resin. The Biogel resin is now ready for use.
[Note: Failure to define the resin results
in 'broken" resin fragments contaminating the final packed chromatography
column. This will slow down the rate of liquid flowing through the column
and increase the time it takes to see the colored proteins separate.]
Student Protocol
In today's exercise, we will
use one of the most popular and ubiquitous methods for the separation of
protein components, size exclusion (gel filtration) column chromatography,
to demonstrate some of the principles of protein purification.
1. Pouring a Biogel size
exclusion column
-
Mount your clean 10 ml pipette
(with top removed) vertically on the column support. Attach a piece of
tubing and clamp to the bottom of the pipette. Close the clamp.
-
Insert a small sample
of glass wool into the column. Using a 1 ml pipette as a "ramrod", position
the glass wool in the bottom of the column to form a small plug.
-
Pipette approximately 5 to 7
ml NAT buffer into the column. Open the bottom clamp (a bit) and allow
the buffer to fill the tubing. Reclamp, leaving approximately 1
to 2 ml NAT buffer in the column.
NOTE: During this time it is vital that you remove air bubbles from
the tubing and the glass wool plug. Any bubbles left in the column will
retard the speed of your column flow.Sometimes this requires tapping
the column and tubing to dislodgetrapped bubbles.
-
Obtain the Biogel resin slurry
to be used in making your column. Gently mix your batch of slurry, then,
with the clamp at the bottom still closed, add slurry to your 10
ml column using a pasteur pipette or other device. Be careful not to introduce
any bubbles into the column. If you see bubbles, use the 1 ml pipette "ramrod"
to stir the slurry in the column, thereby removing them.
-
Allow the column to pack, by
gravity,
with the clamp closed, for 2 to 3 minutes.
-
Place a waste beaker (50 ml)
under the bottom tubing, open the clamp and allow the column to pack. DO
NOT ALLOW THE COLUMN TO RUN DRY. That is, do not allow all of the liquid
above your resin to run out of the column. When necessary, close the clamp,
or continue to add more slurry or buffer to the column, to prevent the
top of the resin from running dry.
As the column packs,
continue to add more mixed slurry to achieve a final packed column height
of ~10 ml. Optimally, the column should be packed without any disruptions
to give a continuous column bed. When adding more slurry to an already
packed column, use the 1 ml "ramrod" to stir up the column bed surface,
then add additional slurry.
2. Washing the column
Wash the packed column
with a few ml of NAT buffer by adding the buffer to the space above the
column. Avoid disturbing the flat, level surface of the top of
the column. Open the bottom clamp to allow the wash buffer to flow
through the column. Collect and discard the eluate in a waste beaker.
AGAIN, DO NOT ALLOW THE COLUMN TO RUN DRY (re-clamp the column
when in danger).
At the end of the wash, clamp
the column off with approximately 1 ml buffer left above the column bed.
3. Separation of the colored
protein standards by column chromatography
Obtain a tube of colored protein
standards.
Number a set of small collection
tubes (1-20) and arrange numerically in a rack. Fill an unlabeled tube
with 0.5 ml of buffer. Note the height of the liquid. Using this tube
as astandard, place a line on the labeled tubes at the 0.5 ml position.
Very carefully, open
the bottom clamp of the column and allow the buffer to drain into a waste
beaker until only approximately 1 millimeter of NAT is left
above the surface of the top of the column. Close clamp.
Position the number 1 tube under
column.
Apply one drop (or 25
m l) of the colored protein standards GENTLY to the column bed surface.
Very carefully, open
clamp slightly and allow the proteins to enter the top of the column bed,
but CLOSE THE CLAMP BEFORE THE TOP OF THE COLUMN GOES DRY.
Gently layer a few drops
of NAT BUFFER on top of column and allow the buffer to enter column as
above. This step makes sure that all of the colored proteins that have
stuck on the column walls have a chance to enter the resin.
Gently fill the column with
buffer to the top, open the clamp, and collect 0.5 ml fractions in tubes
1-20.
[Note: During this process,
a lab partner should be assigned to watch the collection tubes and make
sure that a new tube is placed in position as each tube (1-20) is filled
to the 0.5 ml line.]
Hopefully, all colored proteins
will be eluted from the column by tube 20. If not, continue to collect
0.5 ml samples until all color has eluted from the column. Close
clamp.
Record tube numbers for each
colored protein:
| Tube number(s) |
Protein (Color) |
Molecular weight |
|
blue dextran (blue) |
>2,000,000 |
|
cytochrome c (orange) |
12,400 |
|
vitamin B12 |
1,300 |
Additional activity (if desired)
-
Prepare a graph of the logarithm
of the molecular weight vs elution volume (or tube number) for the three
colored proteins.
-
Predict what tube number a protein
with a molecular weight of 9,000 might elute.
Final Note: The Biogel column
may be poured and stored in the refrigerator for several days before use.
Be sure to tightly clamp the bottom tubing, fill the column with NAT buffer,
and tightly cover the top with plastic wrap to ensure that the column does
not dry out. Do not keep the columns for greater than one week as molds
will eventually grow in the resin. |
