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Molecular Techniques

Protocols for Week 3

Gel Filtration Column Chromatography

I. Calibrating the column

A. Before we use our columns to separate a protein from a complex mixture of proteins, the
columns need to be calibrated. This involves applying a mixture of purified proteins of
varying known molecular masses to the column and subjecting them to the gel filtration
process. This will tell us (i) if the matrix is packed properly, (ii) if the proteins separate
into discrete peaks, and (iii) the void volume.
1. The proteins that you will use to calibrate the column are listed in Table 1.

Table 1. Proteins used to characterize a P30M gel filtration column.


Protein Molecular Mass Amount/ml Color
Ferritin1 445 kDa 4 mg (39.2 µl/ml) yellow/brown
Carbonic Anhydrase2 29 kDa 3 mg none
Cytochrome C3 12.5 kDa 3 mg orange/red
Aprotinin 2,4 6.5 kDa 1-1.5 mg none
1 Ferritin comes already dissolved in buffer at 102 mg/ml and is stored at 4°C.
2 Store at 4°C.
3 Store at –20°C.
4 As a general rule, as molecular mass of a protein decreases, so does solubility in 4 M salt solutions.

Hence, aprotinin is only soluble at about 1-1.5 mg/ml if it is added to basal salts and then gently warmed.
Once dissolved, the solution is then filtered to remove any undissolved aprotinin particulates, and then
the rest of the proteins are added.

2. Refrigerated or frozen vials containing carbonic anhydrase, cytochrome C and


aprotinin are allowed to reach room temperature in a dessicator prior to weighing.
This prevents water from condensing inside the vial and onto the proteins when the
vials are opened. Appropriate amounts of the 3 proteins are weighed out and added to
a screw-capped centrifuge tube. For each milliliter of protein standards, the proteins
are dissolved in 961 µl basal salts and then 39.2 µl of ferritin is added. The final protein
concentration of the standards is 11-11.5 mg/ml (12 mg/ml is the maximum
concentration of proteins to be loaded onto the column).

B. Applying standards to the column


1. The columns have been equilibrating at 0.02 ml/min since last week. Stop the pump
and close the stopcock at the entrance to the column. Carefully untwist the yellow top
and allow it to rest on the reservoir shelf. Reset the pump for the maximum flow rate
of the column (0.30 ml/min) and restart the pump.
2. Allow the buffer on top of the matrix to lower until it is exactly at the level of the
packed matrix. Stop the pump and add 1 ml of your protein standards. To do this, use
a 1 ml pipet and a pipet pump. Fill the pipet with the standards being careful not to

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introduce air bubbles. Apply the standards to the top of the matrix by “necklacing”
them down the side of the column. Keep your pipet tip 1-2 cm from the top of the bed
as you work your way around the edge of the column. Again, be careful not to
introduce air bubbles.
3. Allow the protein mixture to drain down the side of the column and settle on top of the
matrix. Restart the pump and run the standards in at 0.30 ml/min [Note: the outlet
tubing to your pump should be placed into a dry 250 ml graduated cylinder just
before you restart the pump. This is critical for determining your void volume].
While the standards are entering the matrix, take a small amount of buffer (<1 ml) and
rinse any remaining protein off the sides of the column using the “necklace” technique.
Once the standards have completely entered the matrix, add another small amount of
buffer and necklace the sides. Do this twice. Once these rinses have entered the
matrix, change the pump speed to 0.02 ml/min and begin adding buffer back to the
column being careful not to disturb the top of the matrix. Add buffer so that there is a
5-7 cm layer above the matrix.
4. Disconnect the yellow column cap from the stopcock. Clean the outside of the column
cap and the inside of the column with a Kimwipe being careful to remove any drops of
buffer or dried salt or column matrix that may be present. Reinstall the column cap
using a slight twist motion.
5. Fill the buffer reservoir with degassed buffer. Clean the rubber stopper and the inside
rim of the reservoir with a Kimwipe and reinstall the top. Attach a syringe barrel to
the stopcock and open the stopcock. Pull on the syringe so that buffer from the
reservoir passes through the tubing and into the syringe. Close the stopcock, remove
the syringe and reattach the stopcock into the leur fitting of the yellow column top.
Open the stopcock and allow buffer from the reservoir to flow to the column. Note
that the first few drops will come quickly, but then the time between each drop will get
longer and longer.
6. Check for leaks.

C. Run the column at 0.02 ml/min until the ferritin band is near the end of the column. This
will take several days. Collect the column effluent in the graduated cylinder until you
switch to the fraction collector.

SDS-PAGE Gels*
(Tris-Tricine “Schägger” Gels)

Reference: Schägger, H., and G. von Jagow. 1987. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal. Biochem. 166: 368-
379.

I. Introduction

You will be running SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
with tricine (instead of glycine) in the cathode buffer. This type of gel is known as a Schägger
gel. We use this gel system because it can handle salt samples as high as 2.2 M NaCl, and is

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very good at resolving proteins with molecular masses of less than 5 kDa. The salt tolerance
allows us to run samples from our gel filtration column without having to desalt them. You
will be running a 3 layer gel with a 4%T, 3%C stacking gel, a 10%T, 3%C spacing gel, and a
16.5%T, 6%C resolving or separating gel (“T” stands for the total percentage of acrylamide
[acrylamide + bisacrylamide], and “C” stands for the percentage of cross linker
[bisacrylamide] in the total).

II. Preparing to run the gel

A. Stock solutions of buffers and acrylamide are prepared as shown in Tables 1 and 2, and
are filter sterilized using a 0.22 µm polyether sulfone (PES) membrane filter (Millipore).
[Note: In their unpolymerized form, acrylamide and bisacrylamide are potent
neurotoxins.] Whenever possible these solutions should be prepared in the hood.
Gloves, lab coat, and goggles should be worn at all times. All solid waste (gloves,
pipettes, plastic centrifuge tubes, etc.) should be disposed of in the hazardous waste bag
in the hood. The work area and all reagent bottles should be wiped down with a
dampened Kimwipe, and the Kimwipe disposed of in the hazardous waste container.
Since oxygen inhibits the polymerization process, care should be taken to not introduce
bubbles into the acrylamide solutions.

Table 1. Buffers for Tris-Tricine SDS-PAGE1


% Serva
Tris Tricine % SDS % Glycerol % β-Mercapto-
Buffer pH Blue G
(M) (M) (w/v) (w/v) ethanol (v/v)
(w/v)
Anode 0.2 — 8.92 — — — —
buffer
Cathode 0.1 0.1 8.253 0.1 — — —
buffer
Gel 3.0 — 8.452 0.3 — — —
buffer
Schägger
sample 0.05 — 6.82 4 12 0.01 2.0
buffer
1 From: Schägger and von Jagow, 1987.
2 Adjusted with HCl.
3 No correction of the pH, which is around 8.25.

Table 2. Acrylamide-bisacrylamide stock solutions1 .


Acrylamide- % Acrylamide (w/v) % Bisacrylamide (w/v)
bisacrylamide mixture 2
49.5% T3 , 3% C4 48 1.5
49.5% T2 , 6% C3 46.5 3.0
1 From Schägger and von Jagow, 1987.
2 Acrylamide and bisacrylamide powders are placed into screw-capped centrifuge tubes and water is
added directly to the tubes.
3 Percentage of total acrylamide (acrylamide + bisacrylamide).

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4 Percentage of bisacrylamide (crosslinker) in the total.

B. The acrylamide gel solutions are then prepared according to Table 3. Tubes containing
the correct mixtures but lacking APS and TEMED will be prepared for you. The
ammonium persulfate (APS) solution and the TEMED, which catalyze the polymerization
reaction, should be added immediately before the gels are poured.

Table 3. Composition of stacking, spacer and resolving/separating gels1,2 .


Stacking gel Spacer gel Resolving gel
(4% T, 3% C) (10% T, 3% C) (16.5% T, 6% C)
49.5% T , 3% C
2 3 1 ml 6.1 ml —
49.5% T , 6% C
2 3 — — 10 ml
Gel buffer 3.1 ml 10 ml 10 ml
Glycerol3 — — 4 g4
Final Volume (qs 12.5 ml 30 ml 30 ml
with water)5
TEMED 10 µl 10 µl 10 µl
10% APS (w/v) 100 µl 100 µl 100 µl
1 From Schägger and von Jagow, 1987.
2 Solutions are mixed in screw-capped centrifuge tubes with very gentle rocking.
3 For solutions containing glycerol, the glycerol was mixed with the gel buffer and water before the

acrylamide stock solution was added.


4 Since glycerol is a very viscous liquid, it is easier to weigh the proper amount into the mixture than to use

a given volume and pipette it.


5 The volumes of TEMED and APS do not count in the final volume.

III. Setting up the CBS vertical gel rig apparatus.

A. If the glass plates are dirty, clean them with 1% (w/v) SDS and a soft brush, then rinse
with hot water. Inspect the plates for any chips or cracks. Rinse the plates with DI
water, then scrub them with 95% ethanol and dry them off with a Kimwipe until no
streaks are visible. Center the backing plate (the glass plate without a notch on the top
edge, and with rounded corners on the bottom edge) on a pipette tip box or culture tube
rack. Place the gel wrap gasket (orange tubing) around the plate with the rolled edge
facing up and the smooth edge facing down. Make sure that the notches on the gasket
are in the middle of the curved ends of the plate. Gently stretch the gel-wrap from the
center bottom around the curved ends and to the top of the plate using your thumbs.

B. Place 1 mm black spacers on the left and right sides of the plate up against the gasket,
with the curved ends at the bottom and the curve facing out (i.e., the curve of the spacers
matches the curve of the backing plate). Make sure that the spacers do not slip
underneath the gel wrap. Place the notched glass plate on top of the backing plate by
lining up the bottom edges and carefully laying it down from bottom to top. Make sure
that the corners of the gasket are smooth and that the gasket is still flush with the plate
to create a good seal. There are two types of clamps, one for attaching the plates to the
gel rig, and the other for casting the gel. The clamps used for casting are “tighter” and

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their maximum jaw opening is smaller than the clamps used to secure the plates to the
rig. Making sure that the edges of the plates are even with each other, use two casting
clamps to hold the bottom edges of the plates together. Stand the glass plates upright,
make sure they are level with the table top, and place 2-3 more casting clamps on each
side. Using a ruler, measure up 15 cm from the bottom of the plates and mark this
distance on the plate with a Sharpie. This will be the top of the resolving gel. Then
measure 2 cm above the first mark and mark the plates as before. This will be the top of
the spacer gel and the bottom of the stacking gel. Stand the gel rig upright on a bench
protector.

IV. Pouring the gel.

A. Make sure that all the required materials are at your bench before you begin to pour the
gel. The 3 acrylamide solutions for the stacking, spacer, and resolving gels should be on
ice. Add 100 µl of APS to each of the 3 tubes using the P-200 Pipetman. Mix in the APS
by very gently rocking the tubes back and forth, avoiding the introduction of air. The
APS is mixed when there are no lines of schlieren remaining. Add the 10 µl of TEMED
to the resolving gel using the P-10 Pipetman. At this point, you must work steadily and
with purpose so that the acrylamide does not polymerize either in the tube or when you
are pouring it (you have plenty of time to pour). Mix the TEMED into the acrylamide by
gently rocking the tube as was done for the APS. As soon as the solution is mixed, tip
the gel plates back at roughly a 60° angle with the notched plate on top. Pour the
resolving solution directly from the tube between the two plates to the level of the 15 cm
mark. Try to maintain a slow steady flow during the pouring. Carefully add a layer of
water a few millimeters thick on top of the acrylamide in the tube. Replace the screw
cap and allow the remaining acrylamide solution to polymerize in a rack on the bench.

B. As soon as the resolving gel has been poured, add 10 µl of TEMED to the spacer gel, and
once again mix gently by rocking the tube. Using a 2 ml pipette and the blue pipette
pump, pipette the spacer gel solution onto the resolving gel (which has not yet
polymerized) to the second mark on the plate (which is 2 cm above the first). Once
again, maintain a slow steady stream and disturb the interface between the two gels as
little as possible. These two layers should co-polymerize within about 15 min. Once the
spacer gel solution has reached the appropriate level, carefully add a layer of water a
few millimeters thick on top of the acrylamide in the tube, and replace the cap on the
tube and set it on the bench in a tube rack. Finally, add enough DDI water to cover the
top of the spacer gel about 2-3 mm in order to limit the amount of oxygen diffusing into
the gel. Because of the higher percentage of cross-linker in the resolving gel, it will
polymerize first. When the material in both centrifuge tubes has solidified, the material
between the glass plates will be polymerized as well. This will take at least 30 minutes.

C. Once the resolving and spacer gels have polymerized, the stacking gel can be poured.
1. First, pour off the water that lies on top of the spacer gel. This can be facilitated by
pouring the water out of one of the corners of the glass plate sandwich into a

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Kimwipe, and allowing the Kimwipe to soak up as much water as possible by
capillary action.
2. The remaining water needs to be removed and the surface of the spacer gel dried
before pouring the stacking gel. This is best accomplished by folding a Kimwipe and
slipping it between the glass plates. Blot the gel surface free of water using care not to
leave any lint or debris behind. This process may take several Kimwipes.
3. Add 10 µl of TEMED to the tube with the stacking gel and mix as before. Using a 2 ml
pipette, pour the stacking gel on top of the polymerized spacer gel until it is even with
the notch in the top plate. Locate the white plastic comb with the number of “teeth”
required for the desired number of lanes. Starting at one side, slowly insert the comb
at an angle into the stacking gel like a drawbridge, making sure not to trap air bubbles
at the bottoms of the teeth or between the teeth. Center the comb in the gel and make
sure it is level. If air bubbles are trapped, the comb can be pulled out and re-inserted.
Do not put the comb all the way down into the gel. Allow approximately 3 mm of the
comb to remain out of the gel. If at this point the gel does not reach the top edge of
the notch, more stacking gel solution can be added. Do not add stacking gel solution
once the stacking gel has polymerized.
4. Mark the bottom of each well on the glass with a Sharpie. The gel will shrink some as
it polymerizes. Once again, carefully add a layer of water a few millimeters thick on
top of the acrylamide in the tube. Set the tube of extra gel material in a tube rack on
the bench until it has polymerized.
5. Once the stacking gel has polymerized, very carefully remove the comb without
disturbing the wells or tearing any teeth. Once the comb has been removed, the well
teeth need to be straightened. This can be done using a gel-loading tip. At this point,
the gel can be used immediately, or wrapped in plastic wrap and stored for up to one
week at 4° C.

V. Preparing the protein samples.

You will be given a tube (on ice) with a solution of three proteins: carbonic anhydrase (29
kDa), cytochrome C (12.5 kDa), and insulin chain B (~3,500 kDa). The concentration of each of
these proteins in the stock solution is about 1 mg/ml (or 1 µg/µl). Your tube will contain a
1:100 dilution of the stock, or ~10 ng/µl. Prepare 4 protein samples by mixing 1 µl, 3 µl, 7 µl
and 10 µl (10, 30, 70 and 100 ng of protein, respectively) with 20 µl of sample buffer in 0.5 ml
microcentrifuge tubes. Boil the samples for 4 minutes in a heat block with the well filled with
water to facilitate heat conduction to the tubes. After boiling, immediately plunge the tubes
into ice water. Once cooled, the samples should be spun briefly in your centrifuge and are
ready to load onto the gel. The colored markers (ultralow range; 20 µl; Sigma) already contain
dye and sample buffer. They do not need to be mixed with sample buffer, but should be
boiled with the other samples.

VI. Assembling the gel rig.

Remove the clips from the gel slab and carefully remove the gasket. Place the plate in the gel
rig with the notched side facing in. The bottom of the plates should be even with the bottom

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of the lower reservoir, and the notched edge of the plate should be flush with the white gasket
around the outside of the top reservoir. Use one white CBS clamp to clamp the gel plate to the
top reservoir on each side. If only one side of the rig is to be used, place the small glass plate
across the other side of the top reservoir flush with the white gasket and clamp it into place the
same way as the gel slab. Fill the top reservoir with cathode buffer until it covers the top of the
gel by about 1 cm, and fill the bottom reservoir(s) with anode solution.

VII. Loading the gel.

Using the small, plastic bulb pipette, gently flush the wells with cathode buffer to remove any
stray acrylamide and diffused SDS in the wells. Using a different gel loading tip for each
sample, pipette the samples into the desired wells with the P-200 Pipetteman. Place the tip of
the gel loading pipette tip into the well and slowly pipette the protein sample into the well.
There should be 2-3 empty lanes between the color markers and the protein samples.

VIII. Running the gel.

Once the samples have been loaded, cover the reservoirs with the appropriate covers. Attach
the negative (black) lead to the cathode reservoir (top), and the positive (red) lead to the anode
reservoir (s) (bottom). Plug the leads into the same colored slots on the power supply. Choose
one side of the power supply to use. If there are two red leads, plug the second into the back
of the first. Select constant voltage on the power supply. Making sure that the dial is turned to
off, turn the power supply on and press DC start. Turn the dial to 40 V. Many small bubbles
should appear in the cathode buffer, and after a while, some larger ones will appear in the
anode buffer. Run the gel at 40V constant voltage until the dye is completely out of the well
and has fully entered the spacer gel (approximately 1 hour for samples without salt, and
approximately 5-7 hours for samples with salt). Once the sample has entered the spacer gel,
increase the voltage to 100V. Run at 100V constant voltage overnight. Because the gels are so
long, the buffer will be exhausted long before any of the samples can run off the gel. Once the
buffer is exhausted, protein migration stops.

*Some of the text in this section was taken from or adopted from laboratory notebook text
written by Abril Perez while a graduate student in the Shand lab (1/97 – 6/00).

Study Questions

• What is the advantage of using 4 M NaCl as the running buffer in your column and not a
low salt buffer?
• If the matrix got contaminated and microorganisms began to grow in the column, what
could you do to get rid of them?
• Given figures of elution profiles, you should be able to diagnose what is wrong with the
column (e.g., sample volume too large; flow rate too high; etc.).
• What is the difference between the “void volume” and the “bed volume?”

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• Why would ferritin (445,000 Da) and ovalbumin (40,000 Da) elute at the same place from a
P30M column?
• Why was the column run at 0.02 ml/min instead of the higher flow rates suggested in the
matrix manual?
• Why were Tricine SDS-PAGE gels used in class instead of the regular Laemmli gel system?
• What are the differences between Tricine gels and Laemmli gels?
• What do acrylamide, bisacrylamide, TEMED and ammonium persulfate do in the
polymerization of a polyacrylamide gel?
• Why is water laid on top of the spacer gel before the stacking gel is poured?
• Why are the acrylamide solutions handled and mixed so gently prior to pouring of the gel?
• What is in the protein sample buffer and what does each component do?
• Why are the protein samples boiled before loading?
• Why is the presence of SDS so critical in SDS-PAGE?
• How are native protein gels different from SDS-PAGE gels? Is there something special about
the markers used in a native gel?

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